Ovr 110 Antibody Compositions and Methods of Use

ABSTRACT

The invention provides isolated anti-head and neck, ovarian, pancreatic, lung, endometrial or breast cancer antigen (Ovr110) antibodies that internalize upon binding to Ovr110 on a mammalian in vivo. The invention also encompasses compositions comprising an anti-Ovr110 antibody and a carrier. These compositions can be provided in an article of manufacture or a kit. Another aspect of the invention is an isolated nucleic acid encoding an anti-Ovr110 antibody, as well as an expression vector comprising the isolated nucleic acid. Also provided are cells that produce the anti-Ovr110 antibodies. The invention encompasses a method of producing the anti-Ovr110 antibodies. Other aspects of the invention are a method of killing an Ovr110-expressing cancer cell by contacting the cancer cell with an anti-Ovr110 antibody and a method of alleviating or treating an Ovr110-expressing cancer in a mammal by administering a therapeutically effective amount of the anti-Ovr110 antibody to the mammal.

This patent application claims the benefit of priority to U.S.Provisional Application Ser. No. 60/642,490, filed Jan. 7, 2005,teachings of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to anti-Ovr110 antibody compositions andmethods of killing Ovr110-expressing head and neck, ovarian, pancreatic,lung or breast cancers cells.

BACKGROUND OF THE INVENTION Head and Neck Cancer

The annual number of new cases of head and neck cancers in the UnitedStates is approximately 40,000, accounting for about 5% of adultmalignancies. Specifically, the American Cancer Society (ACS; website iscancer with the extension .org of the world wide web) estimates therewill be 29,370, 14,520 and 9,880 new cases of oral cavity and pharynxcancer, esophageal cancer and larynx cancer in 2005, respectively.Furthermore, the ACS estimates there will be 7,320, 13,570 and 3,770deaths from oral cavity and pharynx cancer, esophageal cancer and larynxcancer in 2005, respectively. Adenoid cystic carcinoma (ACC) accountsfor 6% of all salivary gland neoplasms and is the most common malignancyof the submandibular and minor salivary glands. It is not uncommon forrecurrences to occur 10-15 years after initial therapy, adverselyaffecting long term prognosis.

There are three main treatment options for head and neck cancer:surgery, radiation and chemotherapy. One of these therapies, or acombination of them, may be used to treat the cancer. Chemotherapy drugswith single-agent activity in this setting include methotrexate,5-fluorouracil, herein after abbreviated as 5FU, cisplatin, paclitaxel,and docetaxel. Combinations of cisplatin and 5FU, carboplatin and 5FU,and cisplatin and paclitaxel are also used.

Surgery, radiation and chemotherapy to the head and neck can cause manyside effects well known in the art. Improvements in head and neck cancertherapy would include better antitumor efficacy, fewer side effects, andlower cost and hospitalization rates, thereby being more amenable topatients.

The instant invention is a response to the need for an alternativetherapy in the treatment of head and neck cancers. Ovr110, a cellsurface glycoprotein, is reported to be involved in the negativeregulation of T cell activation. Recent studies have shown that Ovr110is also over-expressed in some common epithelial malignancies, includingbreast and ovarian cancer. However, the expression of Ovr110 has notbeen demonstrated in adenoid cystic carcinoma (ACC). The expression ofOvr110 in head and neck cancer is useful as a diagnostic and/ortherapeutic target for head and neck cancer, adenoid cystic carcinoma orother tumors of the salivary glands.

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of head and neck cancer are of critical importance to theoutcome of the patient. Moreover, current procedures, while helpful ineach of these analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop head andneck cancer, for diagnosing head and neck cancer, for monitoring theprogression of the disease, for staging the head and neck cancer, fordetermining whether the head and neck cancer has metastasized, and forimaging the head and neck cancer. There is also a need for bettertreatment of head and neck cancer.

Furthermore, the present invention provides alternative methods oftreating head and neck cancer that overcome the limitations ofconventional therapeutic methods as well as offer additional advantagesthat will be apparent from the detailed description below.

Ovarian Cancer

Cancer of the ovaries is the fourth-most common cause of cancer death inwomen in the United States, with more than 23,000 new cases and roughly14,000 deaths predicted for the year 2001. Shridhar, V. et al., CancerRes. 61(15): 5895-904 (2001); Memarzadeh, S, and Berek, J. S., J.Reprod. Med. 46(7): 621-29 (2001). The ACS estimates that there will beabout 25,580 new cases of ovarian cancer in 2004 and ovarian cancer willcause about 16,090 deaths in the United States. See ACS Website: cancerwith the extension .org of the world wide web. More women die annuallyfrom ovarian cancer than from all other gynecologic malignanciescombined. The incidence of ovarian cancer in the United States isestimated to be 14.2 per 100,000 women per year and 9 women per 100,000die every year from ovarian cancer. In 2004, approximately 70-75% of newdiagnoses will be stage III and IV carcinoma with a predicted 5-yearsurvival of ˜15%. Jemal et al., Annual Report to the Nation on theStatus of Cancer, 1975-2001, with a Special Feature Regarding Survival.Cancer 2004; 101: 3-27. The incidence of ovarian cancer is of seriousconcern worldwide, with an estimated 191,000 new cases predictedannually. Runnebaum, I. B. and Stickeler, E., J. Cancer Res. Clin.Oncol. 127(2): 73-79 (2001). Unfortunately, women with ovarian cancerare typically asymptomatic until the disease has metastasized. Becauseeffective screening for ovarian cancer is not available, roughly 70% ofwomen diagnosed have an advanced stage of the cancer with a five-yearsurvival rate of ˜25-30%. Memarzadeh, S, and Berek, J. S., supra; Nunns,D. et al., Obstet. Gynecol. Surv. 55(12): 746-51. Conversely, womendiagnosed with early stage ovarian cancer enjoy considerably highersurvival rates. Werness, B. A. and Eltabbakh, G. H., Int'l. J. Gynecol.Pathol. 20(1): 48-63 (2001). Although our understanding of the etiologyof ovarian cancer is incomplete, the results of extensive research inthis area point to a combination of age, genetics, reproductive, anddietary/environmental factors. Age is a key risk factor in thedevelopment of ovarian cancer: while the risk for developing ovariancancer before the age of 30 is slim, the incidence of ovarian cancerrises linearly between ages 30 to 50, increasing at a slower ratethereafter, with the highest incidence being among septagenarian women.Jeanne M. Schilder et al., Hereditary Ovarian Cancer: Clinical Syndromesand Management, in Ovarian Cancer 182 (Stephen C. Rubin and Gregory P.Sutton eds., 2d ed. 2001).

With respect to genetic factors, a family history of ovarian cancer isthe most significant risk factor in the development of the disease, withthat risk depending on the number of affected family members, the degreeof their relationship to the woman, and which particular first degreerelatives are affected by the disease. Id. Mutations in several geneshave been associated with ovarian cancer, including BRCA1 and BRCA2,both of which play a key role in the development of breast cancer, aswell as hMSH2 and hMLH1, both of which are associated with hereditarynon-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology,and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (StephenC. Rubin and Gregory P. Sutton eds., 2d ed. 2001). BRCA1, located onchromosome 17, and BRCA2, located on chromosome 13, are tumor suppressorgenes implicated in DNA repair; mutations in these genes are linked toroughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at185-86. bMSH2 and hMLH1 are associated with DNA mismatch repair, and arelocated on chromosomes 2 and 3, respectively; it has been reported thatroughly 3% of heriditary ovarian carcinomas are due to mutations inthese genes. Look, supra at 173; Schilder et al., supra at 184, 188-89.

Reproductive factors have also been associated with an increased orreduced risk of ovarian cancer. Late menopause, nulliparity, and earlyage at menarche have all been linked with an elevated risk of ovariancancer. Schilder et al., supra at 182. One theory hypothesizes thatthese factors increase the number of ovulatory cycles over the course ofa woman's life, leading to “incessant ovulation,” which is thought to bethe primary cause of mutations to the ovarian epithelium. Id.; Laura J.Havrilesky and Andrew Berchuck, Molecular Alterations in SporadicOvarian Cancer, in Ovarian Cancer 25 (Stephen C. Rubin and Gregory P.Sutton eds., 2d ed. 2001). The mutations may be explained by the factthat ovulation results in the destruction and repair of that epithelium,necessitating increased cell division, thereby increasing thepossibility that an undetected mutation will occur. Id. Support for thistheory may be found in the fact that pregnancy, lactation, and the useof oral contraceptives, all of which suppress ovulation, confer aprotective effect with respect to developing ovarian cancer. Id. Amongdietary/environmental factors, there would appear to be an associationbetween high intake of animal fat or red meat and ovarian cancer, whilethe antioxidant Vitamin A, which prevents free radical formation andalso assists in maintaining normal cellular differentiation, may offer aprotective effect. Look, supra at 169. Reports have also associatedasbestos and hydrous magnesium trisilicate (talc), the latter of whichmay be present in diaphragms and sanitary napkins, with ovarian cancer.Id. at 169-70.

Current screening procedures for ovarian cancer, while of some utility,are quite limited in their diagnostic ability, a problem that isparticularly acute at early stages of cancer progression when thedisease is typically asymptomatic yet is most readily treated. Walter J.Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998);Memarzadeh and Berek, supra; Runnebaum and Stickeler, supra; Wemess andEltabbakh, supra. Commonly used screening tests include biannualrectovaginal pelvic examination, radioimmunoassay to detect the CA-125serum tumor marker, and transvaginal ultrasonography. Burdette, supra at166. Currently, CA-125 is the only clinically approved serum marker foruse in ovarian cancer. CA-125 is found elevated in the majority ofserous cancers, but is elevated in only half of those women with earlystage disease. The major clinical application of CA125 is in monitoringtreatment success or detection of recurrence in women undergoingtreatment for ovarian cancer. Markman M. The Oncologist; 2: 6-9 (1997).The use of CA125 as a screening marker is limited because it isfrequently elevated in women with benign diseases such as endometriosis.Hence, there is a critical need for novel serum markers that are moresensitive and specific for the detection of ovarian cancer when usedalone, or in combination with CA125. Bast R C. Et al., Early Detectionof Ovarian Cancer: Promise and Reality in Ovarian Cancer. CancerResearch and Treatment Vol 107 (Stack M S, Fishman, D A, eds., 2001).

Pelvic examination has failed to yield adequate numbers of earlydiagnoses, and the other methods are not sufficiently accurate. Id. Onestudy reported that only 15% of patients who suffered from ovariancancer were diagnosed with the disease at the time of their pelvicexamination. Look, supra at 174. Moreover, the CA-125 test is prone togiving false positives in pre-menopausal women and has been reported tobe of low predictive value in post-menopausal women. Id. at 174-75.Although transvaginal ultrasonography is now the preferred procedure forscreening for ovarian cancer, it is unable to distinguish reliablybetween benign and malignant tumors, and also cannot locate primaryperitoneal malignancies or ovarian cancer if the ovary size is normal.Schilder et al., supra at 194-95. While genetic testing for mutations ofthe BRCA1, BRCA2, hMSH2, and hMLH1 genes is now available, these testsmay be too costly for some patients and may also yield false negative orindeterminate results. Schilder et al., supra at 191-94.

Additionally, current efforts focus on the identification of panels ofbiomarkers that can be used in combination. Bast R C Jr., J Clin Oncol2003; 21: 200-205. Currently, other markers being evaluated as potentialovarian serum markers which may serve as members of a multi-marker panelto improve detection of ovarian cancer are HE4; mesothelin; kallikrein5, 8, 10 and 11; and prostasin. Urban et al. Ovarian cancer screeningHematol Oncol Clin North Am. 2003 August; 17(4):989-1005; Hellstrom etal. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma, CancerRes. 2003 Jul. 1; 63(13):3695-700; Ordonez, Application of mesothelinimmunostaining in tumor diagnosis, Am J Surg Pathol. 2003 November;27(11):1418-28; Diamandis E P et al., Cancer Research 2002; 62: 295-300;Yousef G M et al., Cancer Research 2003; 63: 3958-3965; Kishi T et al.,Cancer Research 2003; 63: 2771-2774; Luo L Y et al., Cancer Research2003; 63: 807-811; Mok S C et al., J Natl Cancer Inst 2001; 93 (19):1437-1439.

The staging of ovarian cancer, which is accomplished through surgicalexploration, is crucial in determining the course of treatment andmanagement of the disease. AJCC Cancer Staging Handbook 187 (Irvin D.Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadehand Berek, supra; Shridhar et al., supra. Staging is performed byreference to the classification system developed by the InternationalFederation of Gynecology and Obstetrics. David H. Moore, PrimarySurgical Management of Early Epithelial Ovarian Carcinoma, in OvarianCancer 203 (Stephen C. Rubin and Gregory P. Sutton eds., 2d ed. 2001);Fleming et al. eds., supra at 188. Stage I ovarian cancer ischaracterized by tumor growth that is limited to the ovaries and iscomprised of three substages. Id. In substage IA, tumor growth islimited to one ovary, there is no tumor on the external surface of theovary, the ovarian capsule is intact, and no malignant cells are presentin ascites or peritoneal washings. Id. Substage IB is identical to IA,except that tumor growth is limited to both ovaries. Id. Substage ICrefers to the presence of tumor growth limited to one or both ovaries,and also includes one or more of the following characteristics: capsulerupture, tumor growth on the surface of one or both ovaries, andmalignant cells present in ascites or peritoneal washings. Id.

Stage II ovarian cancer refers to tumor growth involving one or bothovaries, along with pelvic extension. Id. Substage IIA involvesextension and/or implants on the uterus and/or fallopian tubes, with nomalignant cells in the ascites or peritoneal washings, while substageIIB involves extension into other pelvic organs and tissues, again withno malignant cells in the ascites or peritoneal washings. Id. SubstageIIC involves pelvic extension as in IIA or IIB, but with malignant cellsin the ascites or peritoneal washings. Id.

Stage III ovarian cancer involves tumor growth in one or both ovaries,with peritoneal metastasis beyond the pelvis confirmed by microscopeand/or metastasis in the regional lymph nodes. Id. Substage IIIA ischaracterized by microscopic peritoneal metastasis outside the pelvis,with substage IIIB involving macroscopic peritoneal metastasis outsidethe pelvis 2 cm or less in greatest dimension. Id. Substage IIIC isidentical to IIIB, except that the metastasis is greater than 2 cm ingreatest dimension and may include regional lymph node metastasis. Id.

Lastly, Stage IV refers to the presence distant metastasis, excludingperitoneal metastasis. Id.

While surgical staging is currently the benchmark for assessing themanagement and treatment of ovarian cancer, it suffers from considerabledrawbacks, including the invasiveness of the procedure, the potentialfor complications, as well as the potential for inaccuracy. Moore, supraat 206-208, 213. In view of these limitations, attention has turned todeveloping alternative staging methodologies through understandingdifferential gene expression in various stages of ovarian cancer and byobtaining various biomarkers to help better assess the progression ofthe disease. Vartiainen, J. et al., Int'l J. Cancer, 95(5): 313-16(2001); Shridhar et al. supra; Baekelandt, M. et al., J. Clin. Oncol.18(22): 3775-81.

The treatment of ovarian cancer typically involves a multiprong attack,with surgical intervention serving as the foundation of treatment.Dennis S. Chi and William J. Hoskins, Primary Surgical Management ofAdvanced Epithelial Ovarian Cancer, in Ovarian Cancer 241 (Stephen C.Rubin and Gregory P. Sutton eds., 2d ed. 2001). For example, in the caseof epithelial ovarian cancer, which accounts for ˜90% of cases ofovarian cancer, treatment typically consists of: (1) cytoreductivesurgery, including total abdominal hysterectomy, bilateralsalpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2)adjuvant chemotherapy with paclitaxel and either cisplatin orcarboplatin. Eltabbakh, G. H. and Awtrey, C. S., Expert Op.Pharmacother. 2(10): 109-24. Despite a clinical response rate of 80% tothe adjuvant therapy, most patients experience tumor recurrence withinthree years of treatment. Id. Certain patients may undergo a secondcytoreductive surgery and/or second-line chemotherapy. Memarzadeh andBerek, supra.

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of ovarian cancer are of critical importance to the outcomeof the patient. Moreover, current procedures, while helpful in each ofthese analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop ovariancancer, for diagnosing ovarian cancer, for monitoring the progression ofthe disease, for staging the ovarian cancer, for determining whether theovarian cancer has metastasized, and for imaging the ovarian cancer.There is also a need for better treatment of ovarian cancer.

Breast Cancer

Breast cancer, also referred to as mammary tumor cancer, is the secondmost common cancer among women, accounting for a third of the cancersdiagnosed in the United States. One in nine women will develop breastcancer in her lifetime and about 192,000 new cases of breast cancer arediagnosed annually with about 42,000 deaths. Bevers, Primary Preventionof Breast Cancer, in Breast Cancer, 20-54 (Kelly K Hunt et al., ed.,2001); Kochanek et al., 49 Nat'l. Vital Statistics Reports 1, 14 (2001).Breast cancer is extremely rare in women younger than 20 and is veryrare in women under 30. The incidence of breast cancer rises with ageand becomes significant by age 50. White Non-Hispanic women have thehighest incidence rate for breast cancer and Korean women have thelowest. Increased prevalence of the genetic mutations BRCA1 and BRCA2that promote breast and other cancers are found in Ashkenazi Jews.African American women have the highest mortality rate for breast canceramong these same groups (31 per 100,000), while Chinese women have thelowest at 11 per 100,000. Although men can get breast cancer, this isextremely rare. In the United States it is estimated there will be217,440 new cases of breast cancer and 40,580 deaths due to breastcancer in 2004. (ACS Website: cancer with the extension org of the worldwide web). With the exception of those cases with associated geneticfactors, precise causes of breast cancer are not known.

In the treatment of breast cancer, there is considerable emphasis ondetection and risk assessment because early and accurate staging ofbreast cancer has a significant impact on survival. For example, breastcancer detected at an early stage (stage T0, discussed below) has afive-year survival rate of 92%. Conversely, if the cancer is notdetected until a late stage (i.e., stage T4 (IV)), the five-yearsurvival rate is reduced to 13%. AJCC Cancer Staging Handbook pp. 164-65(Irvin D. Fleming et al. eds., 5^(th) ed. 1998). Some detectiontechniques, such as mammography and biopsy, involve increaseddiscomfort, expense, and/or radiation, and are prescribed only topatients with an increased risk of breast cancer.

Current methods for predicting or detecting breast cancer risk are notoptimal. One method for predicting the relative risk of breast cancer isby examining a patient's risk factors and pursuing aggressive diagnosticand treatment regimens for high risk patients. A patient's risk ofbreast cancer has been positively associated with increasing age,nulliparity, family history of breast cancer, personal history of breastcancer, early menarche, late menopause, late age of first full termpregnancy, prior proliferative breast disease, irradiation of the breastat an early age and a personal history of malignancy. Lifestyle factorssuch as fat consumption, alcohol consumption, education, andsocioeconomic status have also been associated with an increasedincidence of breast cancer although a direct cause and effectrelationship has not been established. While these risk factors arestatistically significant, their weak association with breast cancerlimits their usefulness. Most women who develop breast cancer have noneof the risk factors listed above, other than the risk that comes withgrowing older. NIH Publication No. 00-1556 (2000).

Current screening methods for detecting cancer, such as breast selfexam, ultrasound, and mammography have drawbacks that reduce theireffectiveness or prevent their widespread adoption. Breast self exams,while useful, are unreliable for the detection of breast cancer in theinitial stages where the tumor is small and difficult to detect bypalpation. Ultrasound measurements require skilled operators at anincreased expense. Mammography, while sensitive, is subject to overdiagnosis in the detection of lesions that have questionable malignantpotential. There is also the fear of the radiation used in mammographybecause prior chest radiation is a factor associated with an increasedincidence of breast cancer.

At this time, there are no adequate methods of breast cancer prevention.The current methods of breast cancer prevention involve prophylacticmastectomy (mastectomy performed before cancer diagnosis) andchemoprevention (chemotherapy before cancer diagnosis) which are drasticmeasures that limit their adoption even among women with increased riskof breast cancer. Bevers, supra.

A number of genetic markers have been associated with breast cancer.Examples of these markers include carcinoembryonic antigen (CEA) (Mughalet al., JAMA 249:1881 (1983)), MUC-1 (Frische and Liu, J. Clin. Ligand22:320 (2000)), HER-2/neu (Haris et al., Proc. Am. Soc. Clin. Oncology15:A96 (1996)), uPA, PAI-1, LPA, LPC, RAK and BRCA (Esteva and Fritsche,Serum and Tissue Markers for Breast Cancer, in Breast Cancer, 286-308(2001)). These markers have problems with limited sensitivity, lowcorrelation, and false negatives which limit their use for initialdiagnosis. For example, while the BRCA1 gene mutation is useful as anindicator of an increased risk for breast cancer, it has limited use incancer diagnosis because only 6.2% of breast cancers are BRCA1 positive.Malone et al., JAMA 279:922 (1998). See also, Mewman et al., JAMA279:915 (1998) (correlation of only 3.3%).

There are four primary classifications of breast cancer varying by thesite of origin and the extent of disease development. Classification I,or ductal carcinoma in situ (DCIS) involves malignant transformation ofductal epithelial cells that remain in their normal position. DCIS is apurely localized disease, incapable of metastasis. Classification II orinvasive ductal carcinoma (IDC) involves malignancy of the ductalepithelial cells breaking through the basal membrane and into thesupporting tissue of the breast. IDC may eventually spread elsewhere inthe body. Classification III or lobular carcinoma in situ (LCIS)involves malignancy arising in a single lobule of the breast that failsto extend through the lobule wall. LCIS generally remains localized.Classification IV or infiltrating lobular carcinoma (ILC) involvesmalignancy arising in a single lobule of the breast and invadingdirectly through the lobule wall into adjacent tissues. By virtue of itsinvasion beyond the lobule wall, ILC may penetrate lymphatics and bloodvessels and spread to distant sites.

For purpose of determining prognosis and treatment, these four breastcancer types have been staged according to the size of the primary tumor(T), the involvement of lymph nodes (N), and the presence of metastasis(M). Although DCIS by definition represents localized stage I disease,the other forms of breast cancer may range from stage II to stage IV.There are additional prognostic factors that further serve to guidesurgical and medical intervention. The most common ones are total numberof lymph nodes involved, ER (estrogen receptor) status, Her2/neureceptor status and histologic grades.

Breast cancers are diagnosed into the appropriate stage categoriesrecognizing that different treatments are more effective for differentstages of cancer. Stage TX indicates that primary tumor cannot beassessed (i.e., tumor was removed or breast tissue was removed). StageT0 is characterized by abnormalities such as hyperplasia but with noevidence of primary tumor. Stage Tis is characterized by carcinoma insitu, intraductal carcinoma, lobular carcinoma in situ, or Paget'sdisease of the nipple with no tumor. Stage T1 (I) is characterized ashaving a tumor of 2 cm or less in the greatest dimension. Within stageT1, Tmic indicates microinvasion of 0.1 cm or less, T1a indicates atumor of between 0.1 to 0.5 cm, T1b indicates a tumor of between 0.5 to1 cm, and T1c indicates tumors of between 1 cm to 2 cm. Stage T2 (II) ischaracterized by tumors from 2 cm to 5 cm in the greatest dimension.Tumors greater than 5 cm in size are classified as stage T3 (III). StageT4 (IV) indicates a tumor of any size with extension to the chest wallor skin. Within stage T4, T4a indicates extension of the tumor to thechest wall, T4b indicates edema or ulceration of the skin of the breastor satellite skin nodules confined to the same breast, T4c indicates acombination of T4a and T4b, and T4d indicates inflammatory carcinoma.AJCC Cancer Staging Handbook pp. 159-70 (Irvin D. Fleming et al. eds.,5^(th) ed. 1998). In addition to standard staging, breast tumors may beclassified according to their estrogen receptor and progesteronereceptor protein status. Fisher et al., Breast Cancer Research andTreatment 7:147 (1986). Additional pathological status, such as HER2/neustatus may also be useful. Thor et al., J. Nat'l. Cancer Inst. 90:1346(1998); Paik et al., J. Nat'l. Cancer Inst. 90:1361 (1998); Hutchins etal., Proc. Am. Soc. Clin. Oncology 17:A2 (1998).; and Simpson et al., J.Clin. Oncology 18:2059 (2000).

In addition to the staging of the primary tumor, breast cancermetastases to regional lymph nodes may be staged. Stage NX indicatesthat the lymph nodes cannot be assessed (e.g., previously removed).Stage N0 indicates no regional lymph node metastasis. Stage N1 indicatesmetastasis to movable ipsilateral axillary lymph nodes. Stage N2indicates metastasis to ipsilateral axillary lymph nodes fixed to oneanother or to other structures. Stage N3 indicates metastasis toipsilateral internal mammary lymph nodes. Id.

Stage determination has potential prognostic value and provides criteriafor designing optimal therapy. Simpson et al., J. Clin. Oncology 18:2059(2000). Generally, pathological staging of breast cancer is preferableto clinical staging because the former gives a more accurate prognosis.However, clinical staging would be preferred if it were as accurate aspathological staging because it does not depend on an invasive procedureto obtain tissue for pathological evaluation. Staging of breast cancerwould be improved by detecting new markers in cells, tissues, or bodilyfluids which could differentiate between different stages of invasion.Progress in this field will allow more rapid and reliable methods fortreating breast cancer patients.

Treatment of breast cancer is generally decided after an accuratestaging of the primary tumor. Primary treatment options include breastconserving therapy (lumpectomy, breast irradiation, and surgical stagingof the axilla), and modified radical mastectomy. Additional treatmentsinclude chemotherapy, regional irradiation, and, in extreme cases,terminating estrogen production by ovarian ablation.

Until recently, the customary treatment for all breast cancer wasmastectomy. Fonseca et al., Annals of Internal Medicine 127:1013 (1997).However, recent data indicate that less radical procedures may beequally effective, in terms of survival, for early stage breast cancer.Fisher et al., J. of Clinical Oncology 16:441 (1998). The treatmentoptions for a patient with early stage breast cancer (i.e., stage Tis)may be breast-sparing surgery followed by localized radiation therapy atthe breast. Alternatively, mastectomy optionally coupled with radiationor breast reconstruction may be employed. These treatment methods areequally effective in the early stages of breast cancer. Patients withstage I and stage II breast cancer require surgery with chemotherapyand/or hormonal therapy. Surgery is of limited use in stage III andstage IV patients. Thus, these patients are better candidates forchemotherapy and radiation therapy with surgery limited to biopsy topermit initial staging or subsequent restaging because cancer is rarelycurative at this stage of the disease. AJCC Cancer Staging Handbook 84,164-65 (Irvin D. Fleming et al. eds., 5^(th) ed. 1998).

In an effort to provide more treatment options to patients, efforts areunderway to define an earlier stage of breast cancer with low recurrencewhich could be treated with lumpectomy without postoperative radiationtreatment. While a number of attempts have been made to classify earlystage breast cancer, no consensus recommendation on postoperativeradiation treatment has been obtained from these studies. Page et al.,Cancer 75:1219 (1995); Fisher et al., Cancer 75:1223 (1995); Silversteinet al., Cancer 77:2267 (1996).

Pancreatic Cancer

Pancreatic cancer is the thirteenth-most common cancer and eighth-mostcommon cause of cancer death worldwide. Donghui Li, MolecularEpidemiology, in Pancreatic Cancer 3 (Douglas B. Evans et al. eds.,2002). In the United States, cancer of the pancreas is the fourth-mostcommon cancer in both males and females, accounting for five percent ofcancer deaths and nearly 30,000 deaths overall. Id. The rates ofpancreatic cancer are higher in men than women and higher inAfrican-Americans as opposed to Caucasians. Id. at 9. The mostsignificant predictor of pancreatic cancer is patient age; amongCaucasians, the age-related incidence of pancreatic cancer increasescontinuously, even through the 85 and older category. Id. at 3.Approximately 80% of cases occur in the age range of 60 to 80, withthose in their 80s experiencing a risk of acquiring the disease 40 timesthat of those in their 40s. Id. Furthermore, the American Cancer Societyestimates that there will be about 31,800 new cases of pancreatic cancerin 2004 in the United States alone. Pancreatic cancer will cause about31,200 deaths in the United States in the same year. See ACS Website:cancer with the extension org of the world wide web. Despite the effortsof researchers and physicians in devising treatments for pancreaticcancer, it remains almost universally fatal. James R. Howe, MolecularMarkers as a Tool for the Early Diagnosis of Pancreatic Cancer, inPancreatic Cancer 29 (Douglas B. Evans et al. eds., 2002).

Aside from age, a number of risk factors for pancreatic cancer have beenidentified, including smoking, diet, occupation, certain medicalconditions, heredity, and molecular biologic. Smoking is the mostimportant risk factor for acquiring the disease, with the link betweensmoking and pancreatic cancer being established in numerous studies. Li,supra at 3. The relative risk amounts to at least 1.5, increasing withthe level of smoking to an outer risk ratio of 10-fold. Id. The nextmost important factor appears to be diet, with increased risk associatedwith animal protein and fat intake, and decreased risk associated withintake of fruits and vegetables. Id. at 3-4. As for particularoccupations, excessive rates of pancreatic cancer have been associatedwith workers in chemistry, coal and gas exploration, the metal industry,leather tanning, textiles, aluminum milling, and transportation. Id. at4. A number of medical conditions have also been associated with anincreased incidence of pancreatic cancer, including diabetes, chronicpancreatitis, gastrectomy, and cholecystectomy, although a cause andeffect relationship between these conditions and pancreatic cancer hasnot been established. Id.

Hereditary genetic factors comprise less than 10% of the pancreaticcancer burden, with associations documented with hereditarypancreatitis, as well as germline mutations in familial cancer syndromegenes such as hMSH2 and hMLH1 (hereditary nonpolyposis colon cancer),p16 (familial atypical multiple mole-melanoma) and BRCA 1/BRCA2 (breastand ovarian cancer). Id. at 3. While no other organ has a higherinherited basis for cancer than the pancreas, researchers have beenunable to pinpoint the particular genetic defect(s) that contribute toone's susceptibility to pancreatic cancer. David H. Berger and WilliamE. Fisher, Inherited Pancreatic Cancer Syndromes, in Pancreatic Cancer73 (Douglas B. Evans et al. eds., 2002).

From the standpoint of molecular biology, research has revealed anassociation between pancreatic cancer and a number of genetic mutations,including the activation of the proto-oncogene K-ras and theinactivation of the tumor suppressor genes p53, p16, and DPC4. Marina E.Jean et al., The Molecular Biology of Pancreatic Cancer, in PancreaticCancer 15 (Douglas B. Evans et al. eds., 2002).

In one study of pancreatic adenocarcinomas, 83% possessed K-rasactivation along with inactivation of p16 and p53. Id. K-ras mutationsare found in 80 to 95% of pancreatic adenocarcinomas, with p53, p16, andDPC4 genes being the must frequently deleted tumor suppressor genes incancer of the pancreas. Howe, supra at 29. Homozygous deletions,hypermethylation, and mutations of the p16 gene have been discovered in85 to 98% of adenocarcinomas of the pancreas. Id. As might be expectedby the role of alterations in the K-ras, p53, p16, and DPC4 genes, lossof regulation of the cell cycle would appear to be key to tumorigenesisin the pancreas, and may explain why this cancer is so aggressive. Jean,supra at 15. Research has also revealed a link between this cancer andabnormal regulation of certain growth factors and growth factorreceptors, as well as an upregulation of matrix metalloproteinases andtumor angiogenesis regulators. Id. Epidermal growth factor, fibroblastgrowth factor, transforming growth factor-β, insulin-like growth factor,hepatocyte growth factor, and vascular endothelial growth factor mayplay various roles in pancreatic cancer, although such roles have notbeen elucidated. Id. at 18-22.

The development of screening techniques to detect the presence ofpancreatic cancer is particularly essential for this deadly cancer, asmost patients fail to present until their pancreatic tumors obstruct thebile duct or induce pain, at which point the tumors have invaded thecapillary and lymphatic vessels that surround the pancreas, Howe, supraat 29; unfortunately, patients with the metastatic form of the diseasetypically survive less than one year after diagnosis, Jean et al., supraat 15. While computed tomography (CT) and endoscopic retrogradecholangiopancreatography (ERCP) may assist in the diagnosis ofsymptomatic patients, there is presently no tool for screening forpancreatic tumors that would permit their early discovery, at whichpoint they might be curable. Howe, supra at 29. Markers such ascarcinoembryonic antigen, and antibodies generated against cell lines ofhuman colonic cancer (CA 19-9 and CA 195), human ovarian cancer (CA125), and human pancreatic cancer (SPAN-1 and DUPAN-2) may be elevatedin the serum of patients with pancreatic cancer, but these markers arenot sufficiently reliable to serve as screening tools due to their lackof specificity and appearance late in the disease. Walter J. Burdette,Cancer: Etiology, Diagnosis, and Treatment 99 (1998); Hasholzner, U. etal., Anticancer Res. 19(4A): 2477-80 (1999).

Due to the present lack of adequate screening methods, physicians areincreasingly turning to techniques which employ methods of molecularbiology as the most promising means for early diagnosis of the disease.Howe, supra at 30. At present, there is no high sensitivity, highspecificity marker that enables the detection of pancreatic cancer inasymptomatic individuals, but several biological markers are underinvestigation. Id. Considerable efforts are currently focusing on K-ras,with researchers devising techniques to screen samples of pancreaticjuice, bile, duodenal juice, or ERCP brushings to detect K-rasmutations. Id. Because the collection of these samples is invasive andnot particularly helpful in screening those who are asymptomatic,researchers have also turned to serum and stool analysis for K-rasmutations, with the former being the most promising, as the latter ishindered by the complexity of the source material. Id. at 35-38, 42.Moreover, because serum levels of the transcription factor protein p53may parallel cancer progression, p53 is likewise being studied as apossible tumor marker. Id. at 37; Jean et al., supra at 17.

Once pancreatic cancer has been diagnosed, treatment decisions are madein reference to the stage of cancer progression. A number of imagingtechniques are employed to stage pancreatic cancer, with computedtomography (CT) being the present method of choice, Harmeet Kaur et al.,Pancreatic Cancer: Radiologic Staging, in Pancreatic Cancer 86 (DouglasB. Evans et al. eds., 2002); Ishiguchi, T. et al.,Hepatogastroenterology 48(40): 923-27 (2001), despite the fact that itfrequently underestimates the extent of the cancer, as small-volumemetastases are often beyond the resolution of CT, H. J. Kim and K. C.Conlon, Laparascopic Staging, in Pancreatic Cancer 15 (Douglas B. Evanset al. eds., 2002). MRI may at some point supplant CT in view of, interalia, its ability to (1) contrast among various tissue, (2) modify pulsesequences to improve visualization of lesions and minimize artifacts,(3) perform imaging while limiting a patient's exposure to ionizingradiation, and (4) visualize vessels without using IV iodinated contrastreagents. Kaur et al., supra at 87. At present, however, MRI has notdemonstrated a clear advantage over CT. Kim and Conlon, supra at 116.

A variety of ultrasonic techniques are also currently employed instaging, including transabdominal ultrasound (TUS), endoscopicultrasound (EUS), and intraoperative ultrasound (IUS), with EUS beingone of the most promising. Kaur et al., supra at 86; Richard A.Erickson, Endoscopic Diagnosis and Staging: Endoscopic Ultrasound,Endoscopic Retrograde Cholangiopancreatography, in Pancreatic Cancer97-106 (Douglas B. Evans et al eds., 2002). These techniques, however,are each limited by a variety of factors: TUS is hindered by gas in thegastrointestinal tract and fat in the peritoneum, EUS requiresconsiderable experience in ultrasonography and endoscopy and may not bewidely available, and IUS can only be used intraoperatively. Kaur etal., supra at 86.

Although in its nascent stages, the search for markers that will assistin staging pancreatic cancer has found some possible leads. For example,research has revealed that two metastasis-suppressing genes, nm23-H1 andKAI1, are differentially expressed depending on the stage of pancreaticcancer, with their expression being upregulated at early stages and downregulated at later stages of the disease. Friess, H. et al., J. Clin.Oncol. 19(9): 2422-32 (2001). Researchers have also focused on geneticlymph node staging, particularly searching for mutations in the K-rasproto-oncogene. Yamada, T. et al., Int'l J. Oncol. 16(6): 1165-71(2000). Likewise, research has identified that the presence of mutatedK-ras sequences in plasma/serum is associated with late stage pancreaticcancer, although the presence of early stage pancreatic cancer can bedetected this way as well. Sorenson, G. D., Clin. Cancer Res. 6(6):2129-37 (2000). A promising staging technique using a multimarkerreverse transcriptase-polymerase chain reaction assay has successfullydistinguished pancreatic cancer stages by assaying blood and tissuesamples for mRNA expression of the following tumor markers: the β-humanchorionic gonadotropin gene, the hepatocyte growth factor receptor genec-met, and the β-1,4-N-acetyl-galactosaminyl-transferase gene. Bilchik,A. et al., Cancer 88(5): 1037-44 (2000).

One classification system commonly used to stage pancreatic cancer isthe TNM system devised by the Union Internationale Contre le Cancer.AJCC Cancer Staging Handbook 3 (Irvin D. Fleming et al. eds., 5^(th) ed.1998). This system is divided into several stages, each of whichevaluates the extent of cancer growth with respect to primary tumor (T),regional lymph nodes (N), and distant metastasis (M). Id.

Stage 0 is characterized by carcinoma in situ (Tis), with no regionallymph node metastasis (N0) and no distant metastasis (M0). Id. at 113.Stages I and II differ from stage 0 only in terms of tumor category:stage I involves a tumor limited only to the pancreas that is either (1)2 cm or less in greatest dimension (T1) or (2) more than 2 cm ingreatest dimension (T2), while stage II involves a tumor that extendsdirectly into the duodenum, bile duct, or peripancreatic tissues (T3).Id. Stage III involves tumor category T1, T2, or T3; regional lymph nodemetastasis (N1), which involves either a single lymph node (pN1a) ormultiple lymph nodes (pN1b); and no distant metastasis (M0). Stage IVAis characterized by tumor extension directly into the stomach, spleen,colon, or adjacent large vessels (T4); any N category; and no distantmetastasis (M0). Lastly, stage IVB is characterized by any T category,any N category, and distant metastasis (M1). Id. Once the cancer hasbeen staged, the only consistently effective treatment for the diseaseis surgery, and with only ten to fifteen percent of patients being ableto undergo potentially curative resection. Jean et al., supra at 15;Fleming et al. eds., supra at 111; William F. Regine, PostoperativeAdjuvant Therapy: Past, Present, and Future Trial Development, inPancreatic Cancer 235 (Douglas B. Evans et al. eds., 2002). Moreover,the five-year survival of those patients undergoing resection is belowtwenty percent. Regine, supra at 235. While chemotherapeutic agents suchas gemcitabine and 5-fluorouracil have shown some effectiveness againstpancreatic carcinomas, the reality is that chemotherapy has shown littleimpact on survival from pancreatic cancer. Burdette, supra at 101.Radiation therapy has provided conflicting results with respect to itsefficacy, id., although radiation in combination with 5-fluorouracil hasshown some promise, Regine, supra at 235.

In view of the failure of conventional techniques at treating pancreaticcancer, a number of novel approaches employing the techniques ofmolecular biology have been investigated. Considerable research has beenperformed in the area of gene therapy, including antisense technology,gene-directed prodrug activation strategies, promoter gene strategies,and oncolytic viral therapies. Eugene A. Choi and Francis R. Spitz,Strategies for Gene Therapy, in Pancreatic Cancer 331 (Douglas B. Evanset al. eds., 2002); Kasuya, H. et al., Hepatogastroenterology 48(40):957-61 (2001). Other recent approaches have focused on the inhibition ofmatrix metalloproteinases, enzymes which facilitate the metastasis andinvasion of tumor cells through their degradation of basement membranes,and their role in peritumoral stromal degradation and angiogenesis.Alexander S. Rosemurgy, II and Mahmudul Haq, Role of MatrixMetalloproteinase Inhibition in the Treatment of Pancreatic Cancer, inPancreatic Cancer 369 (Douglas B. Evans et al. eds., 2002).

Angiogenesis in Cancer

Growth and metastasis of solid tumors are also dependent onangiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman,J., 1989, Journal of the National Cancer Institute, 82, 4-6. It has beenshown, for example, that tumors which enlarge to greater than 2 mm mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. Once these new blood vessels become embedded inthe tumor, they provide a means for tumor cells to enter the circulationand metastasize to distant sites such as liver, lung or bone. Weidner,N., et al., 1991, The New England Journal of Medicine, 324(1), 1-8.

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. The process is distinct from vasculogenesis, inthat the new endothelial cells lining the vessel arise fromproliferation of existing cells, rather than differentiating from stemcells. The process is invasive and dependent upon proteolysis of theextracellular matrix (ECM), migration of new endothelial cells, andsynthesis of new matrix components. Angiogenesis occurs duringembryogenic development of the circulatory system; however, in adulthumans, angiogenesis only occurs as a response to a pathologicalcondition (except during the reproductive cycle in women).

Under normal physiological conditions in adults, angiogenesis takesplace only in very restricted situations such as hair growth andwounding healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther.63(3):265-3 11; Ribatti et al., 1991, Haematologica 76(4):3 11-20;Risau, 1997, Nature 386(6626):67 1-4. Angiogenesis progresses by astimulus which results in the formation of a migrating column ofendothelial cells. Proteolytic activity is focused at the advancing tipof this “vascular sprout”, which breaks down the ECM sufficiently topermit the column of cells to infiltrate and migrate. Behind theadvancing front, the endothelial cells differentiate and begin to adhereto each other, thus forming a new basement membrane. The cells thencease proliferation and finally define a lumen for the new arteriole orcapillary.

Unregulated angiogenesis has gradually been recognized to be responsiblefor a wide range of disorders, including, but not limited to, cancer,cardiovascular disease, rheumatoid arthritis, psoriasis and diabeticretinopathy. Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999,Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2):158-64.

Of particular interest is the observation that angiogenesis is requiredby solid tumors for their growth and metastases. Folkman, 1986 supra;Folkman 1990, J. Natl. Cancer list., 82(1) 4-6; Folkman, 1992, SeminCancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24. A tumorusually begins as a single aberrant cell which can proliferate only to asize of a few cubic millimeters due to the distance from availablecapillary beds, and it can stay ‘dormant’ without further growth anddissemination for a long period of time. Some tumor cells then switch tothe angiogenic phenotype to activate endothelial cells, whichproliferate and mature into new capillary blood vessels. These newlyformed blood vessels not only allow for continued growth of the primarytumor, but also for the dissemination and recolonization of metastatictumor cells. The precise mechanisms that control the angiogenic switchare not well understood, but it is believed that neovascularization oftumor mass results from the net balance of a multitude of angiogenesisstimulators and inhibitors Folkman, 1995, supra.

One of the most potent inhibitors of angiogenesis is endostatinidentified by O'Reilly and Folkman. O'Reilly et al., 1997, Cell88(2):277-85; O'Reilly et al., 1994, Cell 79(2):3 15-28. Its discoverywas based on the phenomenon that certain primary tumors can inhibit thegrowth of distant metastases. O'Reilly and Folkman hypothesized that aprimary tumor initiates angiogenesis by generating angiogenicstimulators in excess of inhibitors. However, angiogenic inhibitors, byvirtue of their longer half life in the circulation, reach the site of asecondary tumor in excess of the stimulators. The net result is thegrowth of primary tumor and inhibition of secondary tumor. Endostatin isone of a growing list of such angiogenesis inhibitors produced byprimary tumors. It is a proteolytic fragment of a larger protein:endostatin is a 20 kDa fragment of collagen XVIII (amino acidH1132-K1315 in murine collagen XVIII). Endostatin has been shown tospecifically inhibit endothelial cell proliferation in vitro and blockangiogenesis in vivo. More importantly, administration of endostatin totumor-bearing mice leads to significant tumor regression, and notoxicity or drug resistance has been observed even after multipletreatment cycles. Boehm et al., 1997, Nature 390(6658):404-407. The factthat endostatin targets genetically stable endothelial cells andinhibits a variety of solid tumors makes it a very attractive candidatefor anticancer therapy. Fidler and Ellis, 1994, Cell 79(2):185-8; Gastlet al., 1997, Oncology 54(3):177-84; Hinsbergh et al., 1999, Ann Oncol10 Suppl 4:60-3. In addition, angiogenesis inhibitors have been shown tobe more effective when combined with radiation and chemotherapeuticagents. Klement, 2000, J. Clin Invest, 105(8) R15-24. Browder, 2000,Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279(5349):377-80;Mauceri et al., 1998, Nature 394(6690):287-91.

As discussed above, each of the methods for diagnosing and staging headand neck, ovarian, pancreatic, or breast cancer as well as methods fordiagnosing and staging lung and endometrial cancer are limited by thetechnology employed. Accordingly, there is need for sensitive molecularand cellular markers for the detection of head and neck, ovarian,pancreatic, endometrial, lung or breast cancer. There is a need formolecular markers for the accurate staging, including clinical andpathological staging, of head and neck, ovarian, pancreatic,endometrial, lung or breast cancers to optimize treatment methods. Inaddition, there is a need for sensitive molecular and cellular markersto monitor the progress of cancer treatments, including markers that candetect recurrence of head and neck, ovarian, pancreatic, endometrial,lung or breast cancers following remission.

The present invention provides alternative methods of treating head andneck, ovarian, pancreatic, lung, endometrial or breast cancer thatovercome the limitations of conventional therapeutic methods as well asoffer additional advantages that will be apparent from the detaileddescription below.

Autoimmune Disease

Immune system cellular activity is controlled by a complex network ofcell surface interactions and associated signaling processes. When acell surface receptor is activated by its ligand a signal is sent to thecell, and, depending upon the signal transduction pathway that isengaged, the signal can be inhibitory or activatory. For many receptorsystems cellular activity is regulated by a balance between activatorysignals and inhibitory signals. In some of these it is known thatpositive signals associated with the engagement of a cell surfacereceptor by its ligand are downmodulated or inhibited by negativesignals sent by the engagement of a different cell surface receptor byits ligand.

The biochemical mechanisms of these positive and negative signalingpathways have been studied for a number of known immune system receptorand ligand interactions. Many receptors that mediate positive signalinghave cytoplasmic tails containing sites of tyrosine phosphatasephosphorylation known as immunoreceptor tyrosine-based activation motifs(ITAM). A common mechanistic pathway for positive signaling involves theactivation of tyrosine kinases which phosphorylate sites on thecytoplasmic domains of the receptors and on other signaling molecules.Once the receptors are phosphorylated, binding sites for signaltransduction molecules are created which initiate the signaling pathwaysand activate the cell. The inhibitory pathways involve receptors havingimmunoreceptor tyrosine based inhibitory motifs (ITIM), which, like theITAMs, are phosphorylated by tyrosine kinases. Receptors having thesemotifs are involved in inhibitory signaling because these motifs providebinding sites for tyrosine phosphatases which block signaling byremoving tyrosine from activated receptors or signal transductionmolecules. While many of the details of the activation and inhibitorymechanisms are unknown, it is clear that functional balance in theimmune system depends upon opposing activatory and inhibitory signals.

One example of immune system activity that is regulated by a balance ofpositive and negative signaling is B cell proliferation. The B cellantigen receptor is a B cell surface immunoglobulin which, when bound toantigen, mediates a positive signal leading to B cell proliferation.However, B cells also express Fc.gamma. RIIb1, a low affinity IgGreceptor. When an antigen is part of an immune complex with solubleimmunoglobulin, the immune complex can bind B cells by engaging both theB cell antigen receptor via the antigen and Fc.gamma. RIIb1 via thesoluble immunoglobulin. Co-engagement of the Fc.gamma. RIIb1 with the Bcell receptor complex downmodulates the activation signal and prevents Bcell proliferation. Fc.gamma. RIIb1 receptors contain ITIM motifs whichare thought to deliver inhibitory signals to B cells via interaction ofthe ITIMs with tyrosine phosphatases upon co-engagement with B cellreceptors.

The cytolytic activity of Natural Killer (NK) cells is another exampleof immune system activity which is regulated by a balance betweenpositive signals that initiate cell function and inhibitory signalswhich prevent the activity. The receptors that activate NK cytotoxicactivity are not fully understood. However, if the target cells expresscell-surface MHC class I antigens for which the NK cell has a specificreceptor, the target cell is protected from NK killing. These specificreceptors, known as Killer Inhibitory Receptors (KIRs) send a negativesignal when engaged by their MHC ligand, downregulating NK cellcytotoxic activity.

KIRs belong to the immunoglobulin superfamily or the C-type lectinfamily (see Lanier et al., Immunology Today 17:86-91, 1996). Known humanNK KIRs are members of the immunoglobulin superfamily and displaydifferences and similarities in their extracellular, transmembrane andcytoplasmic regions. A cytoplasmic domain amino acid sequence common tomany of the KIRs is an ITIM motif having the sequence YxxL/V. In somecases, it has been shown that phosphorylated ITIMs recruit tyrosinephosphatases which dephosphorylate molecules in the signal transductionpathway and prevent cell activation (see Burshtyn et al., Immunity4:77-85, 1996). The KIRs commonly have two of these motifs spaced apartby 26 amino acids [YxxL/V(x).sub.26YxxL/V]. At least two NK cellreceptors, each specific for a human leukocyte antigen (HLA) C allele(an MHC class I molecule), exist as an inhibitory and an activatoryreceptor. These receptors are highly homologous in the extracellularportions, but have major differences in their transmembrane andcytoplasmic portions. One of the differences is the appearance of theITIM motif in the inhibitory receptor and the lack of the ITIM motif inthe activating receptor (see Biassoni et al., Journal. Exp. Med,183:645-650, 1996).

An immunoreceptor expressed by mouse mast cells, gp49B1, also a memberof the immunoglobulin superfamily, is known to downregulate cellactivation signals and contains a pair of ITIM motifs. gp49B1 shares ahigh degree of homology with human KIRs (Katz et al., Cell Biology, 93:10809-10814, 1996). Mouse NK cells also express a family ofimmunoreceptors, the Ly49 family, which contain the ITIM motif andfunction in a manner similar to human KIRs. However, the Ly49immunoreceptors have no structural homology with human KIRs and containan extracellular C-type lectin domain, making them a member of thelectin superfamily of molecules (see Lanier et al., Immunology Today17:86-91, 1996).

Clearly, the immune system activatory and inhibitory signals mediated byopposing kinases and phosphatases are very important for maintainingbalance in the immune system. Systems with a predominance of activatorysignals will lead to autoimmunity and inflammation. Immune systems witha predominance of inhibitory signals are less able to challenge infectedcells or cancer cells. Isolating new activatory or inhibitory receptorsis highly desirable for studying the biological signal(s) transduced viathe receptor. Additionally, identifying such molecules provides a meansof regulating and treating diseased states associated with autoimmunity,inflammation and infection.

For example engaging a ligand such as Ovr110 that interacts with a cellsurface receptor having ITIM motifs with an antagonistic antibody orsoluble receptor can be used to activate the specific immune function indisease states associated with suppressed immune function. On the otherhand, using an antagonistic antibody specific to Ovr110 or a solubleform of the Ovr110 receptor can be used to block the interaction ofOvr110 with the cell surface receptor to reduce the specific immunefunction in disease states associated with increased immune function.Conversely, since receptors lacking the ITIM motif send activatorysignals once engaged as described above, the effect of antibodies andsoluble receptors is the opposite of that just described.

As discussed above, methods for diagnosing and staging autoimmunediseases is limited by the technology employed. Accordingly, there isneed for sensitive molecular and cellular markers for the detection ofautoimmune diseases. There is a need for molecular markers for theaccurate staging, including clinical and pathological staging, ofautoimmune diseases to optimize treatment methods. In addition, there isa need for sensitive molecular and cellular markers to monitor theprogress of autoimmune disease treatments, including markers that candetect recurrence of autoimmune diseases following remission.

The present invention provides alternative methods of treatingautoimmune diseases that overcome the limitations of conventionaltherapeutic methods as well as offer additional advantages that will beapparent from the detailed description below.

SUMMARY OF THE INVENTION

This invention is directed to an isolated Ovr110 antibody that binds toOvr110 on a mammalian cell in vivo. The invention is further directed toan isolated Ovr110 antibody that internalizes upon binding to Ovr110 ona mammalian cell in vivo. The antibody may be a monoclonal antibody.Alternatively, the antibody is an antibody fragment or a chimeric or ahumanized antibody. The monoclonal antibody may be produced by ahybridoma selected from the group of hybridomas deposited under AmericanType Culture Collection accession number PTA-5180, PTA-5855, PTA-5856,PTA-5884, PTA-6266, PTA-7128 and PTA-7129.

Alternatively, the antibody may compete for binding to the same epitopeas the epitope bound by the monoclonal antibody produced by a hybridomaselected from the group of hybridomas deposited under the American TypeCulture Collection accession number PTA-5180, PTA-5855, PTA-5856,PTA-5884, PTA-6266, PTA-7128 and PTA-7129.

The invention is also directed to conjugated antibodies. They may beconjugated to a growth inhibitory agent or a cytotoxic agent. Thecytotoxic agent may be selected from the group consisting of toxins,antibiotics, radioactive isotopes and nucleolytic enzymes and toxins.Examples of toxins include, but are not limited to, maytansin,maytansinoids, saporin, gelonin, ricin or calicheamicin.

The antibody may be produced in bacteria. Alternatively, the antibodymay be a humanized form of an anti-Ovr110 antibody produced by ahybridoma selected from the group of hybridomas having ATCC accessionnumber PTA-5180, PTA-5855, PTA-5856, PTA-5884, PTA-6266, PTA-7128 andPTA-7129.

The mammalian cell may be a cancer cell. Preferably, the anti-Ovr110monoclonal antibody inhibits the growth of Ovr110-expressing cancercells in vivo. Preferably, the cancer is selected from the groupconsisting of head and neck, ovarian, pancreatic, lung, endometrial andbreast cancer.

The invention is also directed to a method of producing the antibodiescomprising culturing an appropriate cell and recovering the antibodyfrom the cell culture.

The invention is also directed to compositions comprising the antibodiesand a carrier. The antibody of the composition may be conjugated to acytotoxic agent. The cytotoxic agent may be a radioactive isotope orother chemotherapeutic agent.

The invention is also directed to a method of killing anOvr110-expressing cancer cell, comprising contacting the cancer cellwith the antibodies of this invention, thereby killing the cancer cell.The cancer cell is preferably selected from the group consisting of headand neck, ovarian, pancreatic, lung, endometrial, and breast cancercells. The head and neck, ovarian, or breast cancer may be head and neckadenoid cystic carcinoma, ovarian serous adenocarcinoma or breastinfiltrating ductal carcinoma, respectively, or metastatic cancer. Thebreast cancer may be HER-2 negative breast cancer.

The invention is also directed to a method of alleviating anOvr110-expressing cancer in a mammal, comprising administering atherapeutically effective amount of the antibodies to the mammal.

In addition, the invention is directed to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody as described herein. The article ofmanufacture may also comprise an additional component, e.g., a packageinsert indicating that the composition can be used to treat head andneck, ovarian, pancreatic, lung, endometrial or breast cancer.

The invention is also directed to a method for modulating the signalingof a negatively signaling immune cell Ovr110-receptor comprising bindingOvr110 with anti-Ovr110 antibody thereby reducing a suppressed immunefunction.

Additionally, the invention is directed to a method for modulating animmune response comprising binding Ovr110 with an anti-Ovr110 antibodythereby reducing a suppressed immune function. The modulation may be anincreased immune response or a reduction of suppression of an immuneresponse. The immune response may be against a cancer cell. The cancercell may be selected from the group consisting of head and neck,ovarian, pancreatic, lung, endometrial and breast cancer. The immuneresponse may be increased numbers of lymphocytes surrounding a tumor,increased infiltration of lymphocytes in a tumor, or increasedactivation of lymphocytes.

The invention is also directed to a method for increasing activation oflymphocytes comprising binding Ovr110 with an anti-Ovr110 antibodythereby reducing suppression of lymphocyte activation. The lymphocytemay be a T cell lymphocyte.

Furthermore, disorders mediated by autoimmune disease associated withfailure of negative signaling by receptors binding Ovr110 todownregulate cell function may be treated by administering atherapeutically effective amount of a soluble form of Ovr110 to apatient afflicted with such a disorder. Disorders mediated by diseasestates associated with suppressed immune function can be treated byadministering a therapeutically effective amount of an antagonisticOvr110 antibody. Conversely, disorders mediated by diseases associatedwith failure of activatory signaling by Ovr110 can be treated byadministering a therapeutically effective amount of a soluble form ofOvr110. Disorders mediated by states associated with autoimmune functioncan be treated by administering a therapeutically effective amount of anantagonistic Ovr110 antibody. Such autoimmune disorders include but arenot limited to: Multiple sclerosis, Myasthenia gravis, Autoimmuneneuropathies such as Guillain-Barré, Autoimmune uveitis, Crohn'sDisease, Ulcerative colitis, Primary biliary cirrhosis, Autoimmunehepatitis, Autoimmune hemolytic anemia, Pernicious anemia, Autoimmunethrombocytopenia, Temporal arteritis, Anti-phospholipid syndrome,Vasculitides such as Wegener's granulomatosis, Behcet's disease,Psoriasis, Dermatitis herpetiformis, Pemphigus vulgaris, Vitiligo, Type1 or immune-mediated diabetes mellitus, Grave's Disease, Hashimoto'sthyroiditis, Autoimmune oophoritis and orchitis, Autoimmune disease ofthe adrenal gland, Rheumatoid arthritis, Systemic lupus erythematosus,Scleroderma, Polymyositis, dermatomyositis, Spondyloarthropathies suchas ankylosing spondylitis and Sjogren's syndrome.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Immunohistochemistry Demonstrating Expression of Ovr110 inAdenoid Cystic Carcinoma (ACC) Samples using mAb Ovr110.A57.1.

FIG. 2: Immunohistochemistry Demonstrating Presence of CD3 Positive Tcells in Adenoid Cystic Carcinoma (ACC) Samples using CD3 Ab.

FIG. 3: Immunohistochemistry Demonstrating Presence of CD8 Positive Tcells in Adenoid Cystic Carcinoma (ACC) Samples using CD8 Ab.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Human “Ovr110” as used herein, refers to a protein of 282 amino acidsthat is expressed on the cell surface as a glycoprotein, whosenucleotide and amino acid sequence sequences are as disclosed in e.g.,WO 00/12758, Cancer specific gene (CSG) Ovr110; WO 99/63088,Membrane-bound protein PRO1291; WO00/36107, Human ovarian carcinomaantigen; WO 02/02624-A2, Human B7-like protein (B7-L); WO 2004/101756,Ovr110, the disclosures of which are hereby expressly incorporated byreference. The amino acids 30-282 are presumably on the cell surface.Ovr110 as used herein include allelic variants and conservativesubstitution mutants of the protein which have Ovr110 biologicalactivity.

Ovr110 is known in the literature as B7x, B7H4, B7S1, B7-H4 or B7h.5.The RefSeq database at the NCBI annotates accession NM_(—)024626 as“Homo sapiens V-set domain containing T cell activation inhibitor 1(VTCN1), mRNA”. This nucleotide and the encoded protein NP_(—)078902.1are given the following summary:

-   -   B7H4 belongs to the B7 family (see CD80; MIM 112203) of        costimulatory proteins. These proteins are expressed on the        surface of antigen-presenting cells and interact with ligands        (e.g., CD28; MIM 186760) on T lymphocytes.[supplied by OMIM].

Recently, a series of three independent publications have identifiedOvr110 in mouse and human as new member of the T-cell B7 family ofco-stimulatory molecules, an important class of molecules that verytightly regulate the activation/inhibition of T-cell function. Prasad etal., B7S1, a novel B7 family member that negatively regulates T cellactivation, Immunity 18:863-73 (2003); Sica et al., B7-H4, a molecule ofthe B7 family, negatively regulates T cell immunity, Immunity 18:849-61(2003); and Zang et al., B7x: a widely expressed B7 family member thatinhibits T cell activation, Proc. Natl. Acad. Sci. USA 100:10388-92(2003). The predicted amino acid sequence of the mouse gene for B7S1(Prasad 2003) was highly homologous to our previously identified Ovr110molecule, and the predicted sequence of the human B7-H4/B7x (Sica 2003;Zang 2003) molecules were identical to Ovr110. Indirectimmunofluorescent analysis by flow cytometry further confirmed thebinding of our Ovr110 monoclonal antibodies to activated T-lymphocytepopulations, as described by these authors. A list of referencesdiscussing Ovr110 are listed below, the disclosure of which are herebyincorporated by reference.

Tringler B, Liu W, Corral L, Torkko K C, Enomoto T, Davidson S, Lucia MS, Heinz D E, Papkoff J, Shroyer K R. B7-H4 overexpression in ovariantumors. Gynecol Oncol. 2006 January; 100(1): 44-52. Ichikawa M, Chen L.Role of B7-H1 and B7-H4 molecules in down-regulating effector phase ofT-cell immunity: novel cancer escaping mechanisms. Front Biosci. 2005Sep. 1; 10: 2856-60. Collins M, Ling V, Carreno B M. The B7 family ofimmune-regulatory ligands. Genome Biol. 2005; 6(6): 223. Epub 2005 May31. Salceda S, Tang T, Kmet M, Munteanu A, Ghosh M, Macina R, Liu W,Pilkington G, Papkoff J. The immunomodulatory protein B7-H4 isoverexpressed in breast and ovarian cancers and promotes epithelial celltransformation. Exp Cell Res. 2005 May 15; 306(1): 128-41. Greenwald RJ, Freeman G J, Sharpe A H. The B7 family revisited. Annu Rev Immunol.2005; 23: 515-48. Review. Tringler B, Zhuo S, Pilkington G, Torkko K C,Singh M, Lucia M S, Heinz D E, Papkoff J, Shroyer K R. B7-h4 is highlyexpressed in ductal and lobular breast cancer. Clin Cancer Res. 2005 Mar1; 11(5): 1842-8. Sedy J R, Gavrieli M, Potter K G, Hurchla M A,Lindsley R C, Hildner K, Scheu S, Pfeffer K, Ware C F, Murphy T L,Murphy K M. B and T lymphocyte attenuator regulates T cell activationthrough interaction with herpesvirus entry mediator. Nat Immunol. 2005January; 6(1): 90-8. Epub 2004 Nov. 28. Loke P, Allison J P. Emergingmechanisms of immune regu- lation: the extended B7 family and regulatoryT cells. Arthritis Res Ther. 2004; 6(5): 208-14. Epub 2004 Aug. 5.Review. Wang S, Chen L. Co-signaling molecules of the B7-CD28 family inpositive and negative regulation of T lympho- cyte responses. MicrobesInfect. 2004 July; 6(8): 759- 66. Review. Choi I H, Zhu G, Sica G L,Strome S E, Cheville J C, Lau J S, Zhu Y, Flies D B, Tamada K, Chen L.Genomic organi- zation and expression analysis of B7-H4, an immune in-hibitory molecule of the B7 family. J Immunol. 2003 Nov. 1; 171(9):4650-4. Carreno B M, Collins M. BTLA: a new inhibitory receptor with aB7-like ligand. Trends Immunol. 2003 October; 24(10): 524-7. Review.Prasad D V, Richards S, Mai X M, Dong C. B7S1, a novel B7 family memberthat negatively regulates T cell activa- tion. Immunity. 2003 June;18(6): 863-73. Sica G L, Choi I H, Zhu G, Tamada K, Wang S D, Tamura H,Chapoval A I, Flies D B, Bajorath J, Chen L. B7-H4, a molecule of the B7family, negatively regulates T cell immunity. Immunity. 2003 June;18(6): 849-61. Watanabe N, Gavrieli M, Sedy J R, Yang J, Fallarino F,Loftin S K, Hurchla M A, Zimmerman N, Sim J, Zang X, Murphy T L, RussellJ H, Allison J P, Murphy K M. BTLA is a lympho- cyte inhibitory receptorwith similarities to CTLA-4 and PD-1. Nat Immunol. 2003 July; 4(7):670-9. Epub 2003 Jun. 8.

Our findings that Ovr110 is expressed in ovarian, pancreatic, head andneck, endometrial and breast cancers make this cell surface antigen anattractive target for immunotherapy of these and possibly other tumortypes.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Preferably, the antibody will be purified (1)to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(VH) followed by three constant domains (CH) for each of the α and γchains and four CH domains for [L and F isotypes. Each 6 L chain has atthe N-terminus, a variable domain (VL) followed by a constant domain(CL) at its other end.

The VL is aligned with the VH and the CL is aligned with the firstconstant domain of the heavy chain (CHI).

Particular amino acid residues are believed to form an interface betweenthe light chain and heavy chain variable domains. The pairing of a VHand VL together forms a single antigen-binding site. For the structureand properties of the different classes of antibodies, see, e.g., Basicand Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Teff andTristram G. Parslow (eds.), Appleton and Lange, Norwalk, Conn., 1994,page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 1-10-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aP-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the P-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (LI), 5056 (L2) and 89-97 (L3) in the VL, and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (113) in the VH; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “plimatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least heavy chain constant domains, CHI, CH2 andCH3. The constant domains may be native sequence constant domains (e.g.human native sequence constant domains) or amino acid sequence variantthereof. Preferably, the intact antibody has one or more effectorfunctions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (VH), and the firstconstant domain of one heavy chain (CHI). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)2 fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CHI domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of 8 Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., antibody) derived from nature. Suchnative sequence polypeptides can be isolated from nature or can beproduced by recombinant or synthetic means. Thus, a native sequencepolypeptide can have the amino acid sequence of a naturally occurringhuman polypeptide, murine polypeptide, or polypeptide from any othermammalian species.

The term “amino acid sequence variant” refers to a polypeptide that hasamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants of Ovr110 willpossess at least about 70% homology with the native sequence Ovr110,preferably, at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90% homology, and most preferably atleast 95%. The amino acid sequence variants can possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one which can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, FcεR1.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. Sequence similarity may be measured by any common sequence analysisalgorithm, such as GAP or BESTFIT or other variation Smith-Watermanalignment. See, T. F. Smith and M. S. Waterman, J. Mol. Biol.147:195-197 (1981) and W. R. Pearson, Genomics 11:635-650 (1991).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

As used herein, an anti-Ovr110 antibody that “internalizes” is one thatis taken up by (i.e., enters) the cell upon binding to Ovr110 on amammalian cell (i.e. cell surface Ovr110). The internalizing antibodywill of course include antibody fragments, human or humanized antibodyand antibody conjugate. For therapeutic applications, internalization invivo is contemplated. The number of antibody molecules internalized willbe sufficient or adequate to kill an Ovr110-expressing cell, especiallyan Ovr110-expressing cancer cell. Depending on the potency of theantibody or antibody conjugate, in some instances, the uptake of asingle antibody molecule into the cell is sufficient to kill the targetcell to which the antibody binds. For example, certain toxins are highlypotent in killing such that internalization of one molecule of the toxinconjugated to the antibody is sufficient to kill the tumor cell.

Whether an anti-Ovr110 antibody internalizes upon binding Ovr110 on amammalian cell can be determined by various assays including thosedescribed in the experimental examples below. For example, to testinternalization in vivo, the test antibody is labeled and introducedinto an animal known to have Ovr110 expressed on the surface of certaincells. The antibody can be radiolabeled or labeled with fluorescent orgold particles, for instance. Animals suitable for this assay include amammal such as a NCR nude mouse that contains a human Ovr110-expressingtumor transplant or xenograft, or a mouse into which cells transfectedwith human Ovr110 have been introduced, or a transgenic mouse expressingthe human Ovr110 transgene. Appropriate controls include animals thatdid not receive the test antibody or that received an unrelatedantibody, and animals that received an antibody to another antigen onthe cells of interest, which antibody is known to be internalized uponbinding to the antigen. The antibody can be administered to the animal,e.g., by intravenous injection. At suitable time intervals, tissuesections of the animal can be prepared using known methods or asdescribed in the experimental examples below, and analyzed by lightmicroscopy or electron microscopy, for internalization as well as thelocation of the internalized antibody in the cell. For internalizationin vitro, the cells can be incubated in tissue culture dishes in thepresence or absence of the relevant antibodies added to the culturemedia and processed for microscopic analysis at desired time points. Thepresence of an internalized, labeled antibody in the cells can bedirectly visualized by microscopy or by autoradiography if radiolabeledantibody is used. Alternatively, in a quantitative biochemical assay, apopulation of cells comprising Ovr110-expressing cells are contacted invitro or in vivo with a radiolabeled test antibody and the cells (ifcontacted in vivo, cells are then isolated after a suitable amount oftime) are treated with a protease or subjected to an acid wash to removeuninternalized antibody on the cell surface. The cells are ground up andthe amount of protease resistant, radioactive counts per minute (cpm)associated with each batch of cells is measured by passing thehomogenate through a scintillation counter. Based on the known specificactivity of the radiolabeled antibody, the number of antibody moleculesinternalized per cell can be deduced from the scintillation counts ofthe ground-up cells. Cells are “contacted” with antibody in vitropreferably in solution form such as by adding the cells to the cellculture media in the culture dish or flask and mixing the antibody wellwith the media to ensure uniform exposure of the cells to the antibody.Instead of adding to the culture media, the cells can be contacted withthe test antibody in an isotonic solution such as PBS in a test tube forthe desired time period. In vivo, the cells are contacted with antibodyby any suitable method of administering the test antibody such as themethods of administration described below when administered to apatient.

The faster the rate of internalization of the antibody upon binding tothe Ovr110-expressing cell in vivo, the faster the desired killing orgrowth inhibitory effect on the target Ovr110-expressing cell can beachieved, e.g., by a cytotoxic immunoconjugate. Preferably, the kineticsof internalization of the anti-Ovr110 antibodies are such that theyfavor rapid killing of the Ovr110-expressing target cell. Therefore, itis desirable that the anti-Ovr110 antibody exhibit a rapid rate ofinternalization preferably, within 24 hours from administration of theantibody in vivo, more preferably within about 12 hours, even morepreferably within about 30 minutes to 1 hour, and most preferably,within about 30 minutes. The present invention provides antibodies thatinternalize as fast as about 15 minutes from the time of introducing theanti-Ovr110 antibody in vivo. The antibody will preferably beinternalized into the cell within a few hours upon binding to Ovr110 onthe cell surface, preferably within 1 hour, even more preferably within15-30 minutes.

To determine if a test antibody can compete for binding to the sameepitope as the epitope bound by the anti-Ovr110 antibodies of thepresent invention including the antibodies produced by the hybridomasdeposited with the ATCC, a cross-blocking assay e.g., a competitiveELISA assay can be performed. In an exemplary competitive ELISA assay,Ovr110-coated wells of a microtiter plate, or Ovr110-coated sepharosebeads, are pre-incubated with or without candidate competing antibodyand then a biotin-labeled anti-Ovr110 antibody of the invention isadded. The amount of labeled anti-Ovr110 antibody bound to the Ovr110antigen in the wells or on the beads is measured using avidin-peroxidaseconjugate and appropriate substrate.

Alternatively, the anti-Ovr110 antibody can be labeled, e.g., with aradioactive or fluorescent label or some other detectable and measurablelabel. The amount of labeled anti-Ovr110 antibody that binds to theantigen will have an inverse correlation to the ability of the candidatecompeting antibody (test antibody) to compete for binding to the sameepitope on the antigen, i.e., the greater the affinity of the testantibody for the same epitope, the less labeled anti-Ovr-110 antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as ananti-Ovr110 antibody of the invention if the candidate competingantibody can block binding of the anti-Ovr110 antibody by at least 20%,preferably by at least 20-50%, even more preferably, by at least 50% ascompared to a control performed in parallel in the absence of thecandidate competing antibody (but may be in the presence of a knownnoncompeting antibody). It will be understood that variations of thisassay can be performed to arrive at the same quantitative value.

An antibody having a “biological characteristic” of a designatedantibody, such as any of the monoclonal antibodies Ovr110.A7.1,Ovr110.A110.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1(previously identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1,Ovr110.A89, Ovr110.A99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1,Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2,Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9,Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.I5, Ovr110.I16,Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1,Ovr110.J2 and Ovr110.J3, is one which possesses one or more of thebiological characteristics of that antibody which distinguish it fromother antibodies that bind to the same antigen, Ovr110.A7.1,Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1(previously identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1,Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1,Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2,Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9,Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.I5, Ovr110.I16,Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1,Ovr110.J2 and Ovr110.J3 will bind the same epitope as that bound byOvr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1,Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1,Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107,Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2,Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9,Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.I15, Ovr110.I16,Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1,Ovr110.J2 and Ovr110.J3 (e.g. which competes for binding or blocksbinding of monoclonal antibody Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1,Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously identified asOvr110 A22.1), Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1,Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2,Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1,Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1,Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1,Ovr110.D9.1, Ovr110.I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6,Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13,Ovr110.I14, Ovr110.I15, Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20,Ovr110.I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3 to Ovr110, beable to target an Ovr110-expressing tumor cell in vivo and mayinternalize upon binding to Ovr110 on a mammalian cell in vivo.Likewise, an antibody with the biological characteristic of theOvr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1,Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1,Ovr110.A87.1, Ovr110.A89, Ovr110.A99.1, Ovr110.A102.1, Ovr110.A107,Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2,Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9,Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.I5, Ovr110.I16,Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1,Ovr110.J2 and Ovr110.J3 antibody will have the same epitope binding,targeting, internalizing, tumor growth inhibitory and cytotoxicproperties of the antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of a native Ovr110 protein disclosedherein. Methods for identifying antagonists of an Ovr110 polypeptide maycomprise contacting an Ovr110 polypeptide or a cell expressing Ovr110 onthe cell surface, with a candidate antagonist antibody and measuring adetectable change in one or more biological activities normallyassociated with the Ovr110 polypeptide.

An “antibody that inhibits the growth of tumor cells expressing Ovr110”or a “growth inhibitory” antibody is one which binds to and results inmeasurable growth inhibition of cancer cells expressing oroverexpressing Ovr110. Preferred growth inhibitory anti-Ovr110antibodies inhibit growth of Ovr110-expressing tumor cells e.g.,ovarian, pancreatic, lung or breast cancer cells) by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g. from about 50% to about 100%) as compared to theappropriate control, the control typically being tumor cells not treatedwith the antibody being tested. Growth inhibition can be measured at anantibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200nM in cell culture, where the growth inhibition is determined 1-10 daysafter exposure of the tumor cells to the antibody. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. The antibody isgrowth inhibitory in vivo if administration of the anti-Ovr110 antibodyat about 1 pg/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses Ovr110. Preferably the cell is atumor cell, e.g. an ovarian, pancreatic, lung or breast cell. Variousmethods are available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcells in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRI1B contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126.330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer, ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996) may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, multiple myeloma and B-cell lymphoma, brain, as well as headand neck cancer, and associated metastases. Examples of head and neckcancers include, but are not limited to, adenoid cystic carcinomaslocalized, for example to the tongue, parotid gland, nose, palate, skin,neck, submandibular gland, glottis, sinus, epiglottis, buccal space,nerves, larynx, mouth, pharynx, or cheek.

An “Ovr110-expressing cell” is a cell which expresses endogenous ortransfected Ovr110 on the cell surface. An “Ovr110-expressing cancer” isa cancer comprising cells that have Ovr110 protein present on the cellsurface. An “Ovr110-expressing cancer” produces sufficient levels ofOvr110 on the surface of cells thereof, such that an anti-Ovr110antibody can bind thereto and have a therapeutic effect with respect tothe cancer. A cancer which “overexpresses” Ovr110 is one which hassignificantly higher levels of Ovr110 at the cell surface thereof,compared to a noncancerous cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. Ovr110 overexpression may be determined ina diagnostic or prognostic assay by evaluating increased levels of theOvr110 protein present on the surface of a cell (e.g. via animmunohistochemistry assay; FACS analysis). Alternatively, oradditionally, one may measure levels of Ovr110-encoding nucleic acid ormRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; seeWO98/45479 published October, 1998), Southern blotting, Northernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). One may also study Ovr110 overexpressionby measuring antigen in a biological fluid such as serum, e.g., usingantibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issuedJun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No.5,401,638 issued Mar. 28, 1995; and Sias et al. J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays, various in vivo assays areavailable to the skilled practitioner. For example, one may expose cellswithin the body of the patient to an antibody which is optionallylabeled with a detectable label, e.g. a radioactive isotope, and bindingof the antibody to cells in the patient can be evaluated, e.g. byexternal scanning for radioactivity or by analyzing a biopsy taken froma patient previously exposed to the antibody. An Ovr110-expressingcancer includes ovarian, pancreatic, lung or breast cancer. Bodilyfluids include all internal, secreted, expelled and derivative fluids ofthe body such as blood, plasma, serum, urine, saliva, sputum, tears,ascites, peritoneal wash fluid, lymphatic fluid, bile, semen, puss,Amniotic fluid, Aqueous humour, Cerumen, Chyle, Chyme, Interstitialfluid, Menses, Milk, Mucus, Pleural fluid, sweat, Vaginal lubrication,vomit, cerebrospinal fluid and synovial fluid.

A “mammal” for purposes of treating a cancer or alleviating the symptomsof cancer, refers to any mammal, including-humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal ishuman.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an Ovr110-expressing cancer if, after receiving atherapeutic amount of an anti-Ovr110 antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer; reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe anti-Ovr110 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of these signsor symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR).

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See preceding definition of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed.

Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, e.g., gelonin,ricin, saporin, and the various antitumor or anticancer agents disclosedbelow. Other cytotoxic agents are described below. A tumoricidal agentcauses destruction of tumor cells.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anOvr110-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of Ovr110-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Label” as used herein refers to a detectable compound or compositionwhich is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term “epitope tagged” used herein refers to a chimeric polypeptidecomprising an anti-Ovr110 antibody polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the Ig polypeptide to whichit is fused. The tag polypeptide is also preferably fairly unique sothat the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated nucleic acid molecule” is a nucleic acid molecule, e.g., aRNA, DNA, or a mixed polymer, which is substantially separated fromother genome DNA sequences as well as proteins or complexes such asribosomes and polymerases, which naturally accompany a native sequence.The term embraces a nucleic acid molecule which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogues or analoguesbiologically synthesized by heterologous systems. A substantially purenucleic acid molecule includes isolated forms of the nucleic acidmolecule.

“Vector” includes shuttle and expression vectors and includes, e.g., aplasmid, cosmid, or phagemid. Typically, a plasmid construct will alsoinclude an origin of replication (e.g., the ColE1 origin of replication)and a selectable marker (e.g., ampicillin or tetracycline resistance),for replication and selection, respectively, of the plasmids inbacteria. An “expression vector” refers to a vector that contains thenecessary control sequences or regulatory elements for expression of theantibodies including antibody fragment of the invention, in prokaryotic,e.g., bacterial, or eukaryotic cells. Suitable vectors are disclosedbelow.

The cell that produces an anti-Ovr110 antibody of the invention willinclude the parent hybridoma cell e.g., the hybridomas that aredeposited with the ATCC, as well as bacterial and eukaryotic host cellsinto which nucleic acid encoding the antibodies have been introduced.Suitable host cells are disclosed below.

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

Short interfering RNA mediated RNAi has been studied in a variety ofsystems. Fire et al., 1998, Nature, 391, 806, were the first to observeRNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70,describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature, 404, 293, describe RNAi in Drosophila cells transfected withdsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashiret al., 2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of a siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 both suggest that siRNA“may include modifications to either the phosphate-sugar back bone orthe nucleoside to include at least one of a nitrogen or sulfurheteroatom”, however neither application teaches to what extent thesemodifications are tolerated in siRNA molecules nor provide any examplesof such modified siRNA. Kreutzer and Limmer, Canadian Patent ApplicationNo. 2,359,180, also describe certain chemical modifications for use indsRNA constructs in order to counteract activation of doublestranded-RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer and Limmer similarly fail to show towhat extent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that “RNAs with two (phosphorothioate) modified bases also hadsubstantial decreases in effectiveness as RNAi triggers (data notshown); (phosphorothioate) modification of more than two residuesgreatly destabilized the RNAs in vitro and we were not able to assayinterference activities.” Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and observed that substituting deoxynucleotides forribonucleotides “produced a substantial decrease in interferenceactivity”, especially in the case of Uridine to Thymidine and/orCytidine to deoxy-Cytidine substitutions. Id. In addition, the authorstested certain base modifications, including substituting 4-thiouracil,5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, andinosine for guanosine in sense and antisense strands of the siRNA, andfound that whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine “produced a substantial decrease in interferenceactivity” when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resultedin substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describesspecific methods for attenuating gene expression using endogenouslyderived dsRNA. Tuschl et al., International PCT Publication No. WO01/75164, describes a Drosophila in vitro RNAi system and the use ofspecific siRNA molecules for certain functional genomic and certaintherapeutic applications; although Tuschl, 2001, Chem. Biochem., 2,239-245, doubts that RNAi can be used to cure genetic diseases or viralinfection due “to the danger of activating interferon response”. Li etal., International PCT Publication No. WO 00/44914, describes the use ofspecific dsRNAs for use in attenuating the expression of certain targetgenes. Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expression ofparticular genes in mammalian cells using certain dsRNA molecules. Fireet al., International PCT Publication No. WO 99/32619, describesparticular methods for introducing certain dsRNA molecules into cellsfor use in inhibiting gene expression. Plaetinck et al., InternationalPCT Publication No. WO 00/01846, describes certain methods foridentifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describes theidentification of specific genes involved in dsRNA mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Driscoll et al.,International PCT Publication No. WO 01/49844, describes specific DNAconstructs for use in facilitating gene silencing in targeted organisms.Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specificchemically modified siRNA constructs targeting the unc-22 gene of C.elegans. Tuschl et al., International PCT Publication No. WO 02/44321,describe certain synthetic siRNA constructs.

Compositions and Methods of the Invention

The invention provides anti-Ovr110 antibodies. Preferably, theanti-Ovr110 antibodies internalize upon binding to cell surface Ovr110on a mammalian cell. The anti-Ovr110 antibodies may also destroy or leadto the destruction of tumor cells bearing Ovr110.

It was not apparent that Ovr110 was internalization-competent. Inaddition the ability of an antibody to internalize depends on severalfactors including the affinity, avidity, and isotype of the antibody,and the epitope that it binds. We have demonstrated herein that the cellsurface Ovr110 is internalization competent upon binding by theanti-Ovr110 antibodies of the invention. Additionally, it wasdemonstrated that the anti-Ovr110 antibodies of the present inventioncan specifically target Ovr110-expressing tumor cells in vivo andinhibit or kill these cells. These in vivo tumor targeting,internalization and growth inhibitory properties of the anti-Ovr110antibodies make these antibodies very suitable for therapeutic uses,e.g., in the treatment of various cancers including ovarian, pancreatic,lung or breast cancer. Internalization of the anti-Ovr110 antibody ispreferred, e.g., if the antibody or antibody conjugate has anintracellular site of action and if the cytotoxic agent conjugated tothe antibody does not readily cross the plasma membrane (e.g., the toxincalicheamicin). Internalization is not necessary if the antibodies orthe agent conjugated to the antibodies do not have intracellular sitesof action, e.g., if the antibody can kill the tumor cell by ADCC or someother mechanism.

The anti-Ovr110 antibodies of the invention also have variousnon-therapeutic applications. The anti-Ovr110 antibodies of the presentinvention can be useful for diagnosis and staging of Ovr110-expressingcancers (e.g., in radioimaging). They may be used alone or incombination with other ovarian cancer markers, including, but notlimited to, CA125, HE4 and mesothelin. The antibodies are also usefulfor purification or immunoprecipitation of Ovr110 from cells, fordetection and quantitation of Ovr110 in vitro, e.g. in an ELISA or aWestern blot, to kill and eliminate Ovr110-expressing cells from apopulation of mixed cells as a step in the purification of other cells.The internalizing anti-Ovr110 antibodies of the invention can be in thedifferent forms encompassed by the definition of “antibody” herein.Thus, the antibodies include full length or intact antibody, antibodyfragments, native sequence antibody or amino acid variants, humanized,chimeric or fusion antibodies, immunoconjugates, and functionalfragments thereof. In fusion antibodies, an antibody sequence is fusedto a heterologous polypeptide sequence. The antibodies can be modifiedin the Fc region to provide desired effector functions. As discussed inmore detail in the sections below, with the appropriate Fc regions, thenaked antibody bound on the cell surface can induce cytotoxicity, e.g.,via antibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate or reduceeffector function, so as to minimize side effects or therapeuticcomplications, certain other Fc regions may be used.

The antibody may compete for binding, or binds substantially to, thesame epitope bound by the antibodies of the invention. Antibodies havingthe biological characteristics of the present anti-Ovr110 antibodies ofthe invention are also contemplated, e.g., an anti-Ovr110 antibody whichhas the biological characteristics of a monoclonal antibody produced bythe hybridomas accorded ATCC accession numbers PTA-5180, PTA-5855,PTA-5856, PTA-5884, PTA-6266, PTA-7128 and PTA-7129, specificallyincluding the in vivo tumor targeting, internalization and any cellproliferation inhibition or cytotoxic characteristics. Specificallyprovided are anti-Ovr110 antibodies that bind to an epitope present inamino acids 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270,270-282 or 21-35, 31-45, 41-55, 51-65, 61-75, 71-85, 81-95, 91-105,101-115, 111-125, 121-135, 131-145, 141-155, 151-165, 161-175, 171-185,181-195, 191-205, 201-215, 211-225, 221-235, 231-245, 241-255, 251-258of human Ovr110.

Methods of producing the above antibodies are described in detail below.

The present anti-Ovr110 antibodies are useful for treating anOvr110-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such cancers include, but are not limited to headand neck, ovarian, pancreatic, lung, endometrial and or breast cancer.The cancers encompass metastatic cancers of any of the preceding, e.g.,head and neck, ovarian, pancreatic, lung, endometrial or breast cancermetastases. The antibody is able to bind to at least a portion of thecancer cells that express Ovr110 in the mammal and preferably is onethat does not induce or that minimizes HAMA response. Preferably, theantibody is effective to destroy or kill Ovr110-expressing tumor cellsor inhibit the growth of such tumor cells, in vitro or in vivo, uponbinding to Ovr110 on the cell. Such an antibody includes a nakedanti-Ovr110 antibody (not conjugated to any agent). Naked anti-Ovr110antibodies having tumor growth inhibition properties in vivo include theantibodies described in the Experimental Examples below. Nakedantibodies that have cytotoxic or cell growth inhibition properties canbe further conjugated with a cytotoxic agent to render them even morepotent in tumor cell destruction. Cytotoxic properties can be conferredto an anti-Ovr110 antibody by, e.g., conjugating the antibody with acytotoxic agent, to form an immunoconjugate as described below. Thecytotoxic agent or a growth inhibitory agent is preferably a smallmolecule. Toxins such as maytansin, maytansinoids, saporin, gelonin,ricin or calicheamicin and analogs or derivatives thereof, arepreferable.

The invention provides a composition comprising an anti-Ovr110 antibodyof the invention, and a carrier. For the purposes of treating cancer,compositions can be administered to the patient in need of suchtreatment, wherein the composition can comprise one or more anti-Ovr110antibodies present as an immunoconjugate or as the naked antibody.Further, the compositions can comprise these antibodies in combinationwith other therapeutic agents such as cytotoxic or growth inhibitoryagents, including chemotherapeutic agents. The invention also providesformulations comprising an anti-Ovr110 antibody of the invention, and acarrier. The formulation may be a therapeutic formulation comprising apharmaceutically acceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding theinternalizing anti-Ovr110 antibodies. Nucleic acids encoding both the Hand L chains and especially the hypervariable region residues, chainswhich encode the native sequence antibody as well as variants,modifications and humanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating anOvr110-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an internalizing anti-Ovr110 antibody to the mammal. Theantibody therapeutic compositions can be administered short term (acute)or chronic, or intermittent as directed by physician. Also provided aremethods of inhibiting the growth of, and killing an Ovr110 expressingcell. Finally, the invention also provides kits and articles ofmanufacture comprising at least one antibody of this invention,preferably at least one internalizing anti-Ovr110 antibody of thisinvention. Kits containing anti-Ovr110 antibodies find use in detectingOvr-110 expression, or in therapeutic or diagnostic assays, e.g., forOvr110 cell killing assays or for purification and/orimmunoprecipitation of Ovr110 from cells. For example, for isolation andpurification of Ovr110, the kit can contain an anti-Ovr110 antibodycoupled to a solid support, e.g., a tissue culture plate or beads (e.g.,sepharose beads). Kits can be provided which contain antibodies fordetection and quantitation of Ovr110 in vitro, e.g. in an ELISA or aWestern blot. Such antibody useful for detection may be provided with alabel such as a fluorescent or radiolabel.

Production of Anti-Ovr110 Antibodies

The following describes exemplary techniques for the production of theantibodies useful in the present invention. Some of these techniques aredescribed further in Example 1. The Ovr110 antigen to be used forproduction of antibodies may be, e.g., the full length polypeptide or aportion thereof, including a soluble form of Ovr110 lacking the membranespanning sequence, or synthetic peptides to selected portions of theprotein.

Alternatively, cells expressing Ovr110 at their cell surface (e.g. CHOor NIH-3T3 cells transformed to overexpress Ovr110; ovarian, pancreatic,lung, breast or other Ovr110-expressing tumor cell line), or membranesprepared from such cells can be used to generate antibodies. Thenucleotide and amino acid sequences of human and murine Ovr110 areavailable as provided above. Ovr110 can be produced recombinantly in andisolated from, prokaryotic cells, e.g., bacterial cells, or eukaryoticcells using standard recombinant DNA methodology. Ovr110 can beexpressed as a tagged (e.g., epitope tag) or other fusion protein tofacilitate its isolation as well as its identification in variousassays.

Antibodies or binding proteins that bind to various tags and fusionsequences are available as elaborated below. Other forms of Ovr110useful for generating antibodies will be apparent to those skilled inthe art.

Tags

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). The FLAG-peptide (Hopp et al., BioTechnology, 6:1204-1210(1988)) is recognized by an anti-FLAG M2 monoclonal antibody (EastmanKodak Co., New Haven, Conn.). Purification of a protein containing theFLAG peptide can be performed by immunoaffinity chromatography using anaffinity matrix comprising the anti-FLAG M2 monoclonal antibodycovalently attached to agarose (Eastman Kodak Co., New Haven, Conn.).Other tag polypeptides include the KT3 epitope peptide [Martin et al.,Science, 255:192-194 (1992)]; an α-tubulin epitope peptide (Skinner etal., J. Biol. Chenz., 266:15163-15166 (1991)); and the T7 gene proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals, preferablynon-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the relevant antigen and an adjuvant. It may be useful toconjugate the relevant antigen (especially when synthetic peptides areused) to a protein that is immunogenic in the species to be immunized.For example, the antigen can be conjugated to keyhole limpet hemocyanin(KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, usinga bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride, SOCl₂, or R¹N═C═NR, where R and R′ are different alkylgroups. Conjugates also can be made in recombinant cell culture asprotein fusions.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 5-100 pg of the protein or conjugate(for rabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with ⅕ to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later, the animals arebled and the serum is assayed for antibody titer. Animals are boosteduntil the titer plateaus. Also, aggregating agents such as alum aresuitably used to enhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as a hamster, isimmunized as described above to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. After immunization, lymphocytes are isolated andthen fused with a “fusion partner”, e.g., a myeloma cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies. Principles and Practice, pp 103(Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, fusion partner,e.g., the parental myeloma cells. For example, if the parental myelomacells lack the enzyme hypoxanthine guanine phosphoribosyl transferase(HGPRT or HPRT), the selective culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-IImouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980). Once hybridoma cells that produce antibodiesof the desired specificity, affinity, and/or activity are identified,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, pp 103 (Academic Press, 1986)). Suitable culture media forthis purpose include, for example, D-MEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal e.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transformed or transfected intoprokaryotic or eukaryotic host cells such as, e.g., E coli cells, simianCOS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, that donot otherwise produce antibody protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) andPhickthun, Immunol. Revs., 130:151-188 (1992).

Further, the monoclonal antibodies or antibody fragments can be isolatedfrom antibody phage libraries generated using the techniques describedin McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (CH and CL) sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenonimmunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is nonhuman. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art.

Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized anti-Ovr110 antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669(all of GenPharm); 5,545,807; and Alternatively, phage displaytechnology (McCafferty et al., Nature 348:552-553 (1990)) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905. As discussed above, human antibodies may alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors. Various techniques have been developed for the productionof antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E coli,thus allowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab)₂ fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice may also be a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.No. 5,587,458. Fv and sFv are the only species with intact combiningsites that are devoid of constant regions; thus, they are suitable forreduced nonspecific binding during in vivo use. sFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the Ovr110 protein. Other suchantibodies may combine an Ovr110 binding site with a binding site foranother protein. Alternatively, an anti-Ovr110.Arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aTcell receptor molecule (e.g. C133), or Fc receptors for IgG (FcγR),such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focusand localize cellular defense mechanisms to the Ovr110-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express Ovr110. These antibodies possess an Ovr110-bindingarm and an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab)₂ bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (CHI) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

Preferably, the bispecific antibodies in this approach are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers.

The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a VH connected to a VL by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1(X1 n-VD2-(X2)n-Fc,wherein VDI is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fcregion chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the anti-Ovr110 antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the anti-Ovr110 antibody areprepared by introducing appropriate nucleotide changes into theanti-Ovr110 antibody nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the anti-Ovr110 antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe anti-Ovr110 antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-Ovr110 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues within the anti-Ovr110 antibody are identified (e.g.,charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with Ovr110antigen.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at a target codon or region and theexpressed anti-Ovr110 antibody variants are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-Ovr110 antibody with an N-terminal methionyl residue or theantibody fused to a cytotoxic polypeptide. Other insertional variants ofthe anti-Ovr110 antibody molecule include the fusion to the N- orC-terminus of the anti-Ovr110 antibody to an enzyme (e.g. for ADEPT) ora fusion to a polypeptide which increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-Ovr110antibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but FR alterations are also contemplated. Conservativesubstitutions are shown in Table I under the heading of “preferredsubstitutions”. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated “exemplarysubstitutions” in Table 1, or as further described below in reference toamino acid classes, may be introduced and the products screened for adesired characteristic.

TABLE I Amino Acid Substitutions Original Exemplary SubstitutionsPreferred Substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln; asnlys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu Leu(L) norleucine; ile; val; met; ala; ile Lys (K) arg; gln; asn arg Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) alaala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp;phe; thr; ser Phe Val (V) ile; leu; met; phe; ala; leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutralhydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin,his, lys, arg; (5) residues that influence chain orientation: gly, pro;and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the anti-Ovr110 antibody also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibody variants thus generated are displayed in a monovalent fashionfrom filamentous phage particles as fusions to the gene III product ofM13 packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human Ovr110. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-Ovr110 antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared nucleic acid molecule encoding a variant or a non-variantversion of the anti-Ovr110 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of the antibody.

Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

The growth inhibitory effects of an anti-Ovr110 antibody of theinvention may be assessed by methods known in the art, e.g., using cellswhich express Ovr110 either endogenously or following transfection withthe Ovr110 gene. For example, the tumor cell lines andOvr110-transfected cells provided in Example 1 below may be treated withan anti-Ovr110 monoclonal antibody of the invention at variousconcentrations for a few days (e.g., 2-7) days and stained with crystalviolet or MTT or analyzed by some other colorimetric assay. Anothermethod of measuring proliferation would be by comparing ³H-thymidineuptake by the cells treated in the presence or absence an anti-Ovr110antibody of the invention. After antibody treatment, the cells areharvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriated positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. Preferably, thetumor cell is one that over-expresses Ovr110. Preferably, theanti-Ovr110 antibody will inhibit cell proliferation of anOvr110-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, more preferably, by about 30-100%,and even more preferably by about 50-100% or 70-100%, at an antibodyconcentration of about 0.5 to 30 μg/ml. Growth inhibition can bemeasured at an antibody concentration of about 0.5 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.The antibody is growth inhibitory in vivo if administration of theanti-Ovr110 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), tryptan blue or7AAD uptake may be assessed relative to a control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.Ovr110-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g., about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on Ovr110 bound by anantibody of interest, e.g., the Ovr110 antibodies of this invention, aroutine cross-blocking assay such as that describe in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if atest antibody binds the same site or epitope as an anti-Ovr110 antibodyof the invention. Alternatively, or additionally, epitope mapping can beperformed by methods known in the art. For example, the antibodysequence can be mutagenized such as by alanine scanning, to identifycontact residues. The mutant antibody is initially tested for bindingwith polyclonal antibody to ensure proper folding. In a differentmethod, peptides corresponding to different regions of Ovr110 can beused in competition assays with the test antibodies or with a testantibody and an antibody with a characterized or known epitope.

For example, a method to screen for antibodies that bind to an epitopewhich is bound by an antibody this invention may comprise combining anOvr110-containing sample with a test antibody and an antibody of thisinvention to form a mixture, the level of Ovr110 antibody bound toOvr110 in the mixture is then determined and compared to the level ofOvr110 antibody bound in the mixture to a control mixture, wherein thelevel of Ovr110 antibody binding to Ovr110 in the mixture as compared tothe control is indicative of the test antibody's binding to an epitopethat is bound by the anti-Ovr110 antibody of this invention. The levelof Ovr110 antibody bound to Ovr110 is determined by ELISA. The controlmay be a positive or negative control or both. For example, the controlmay be a mixture of Ovr110, Ovr110 antibody of this invention and anantibody known to bind the epitope bound by the Ovr110 antibody of thisinvention. The anti-Ovr110 antibody labeled with a label such as thosedisclosed herein. The Ovr110 may be bound to a solid support, e.g., atissue culture plate or to beads, e.g., sepharose beads.

Immunoconjugates

The invention also pertains to therapy with immunoconjugates comprisingan antibody conjugated to an anti-cancer agent such as a cytotoxic agentor a growth inhibitory agent.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin,maytansinoids, a trichothene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

Preferably, an anti-Ovr110 antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the cast Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DMI linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al. CancerResearch 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA. 1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA. 1-maytansonoid conjugate wastested in vitro on the human breast cancer cell line SK-BR-3, whichexpresses 3×10 5 HER-2 surface antigens per cell. The drug conjugateachieved a degree of cytotoxicity similar to the free maytansonoid drug,which could be increased by increasing the number of maytansinoidmolecules per antibody molecule. The A7-maytansinoid conjugate showedlow systemic cytotoxicity in mice.

Anti-Ovr110 Antibody-Maytanisinoid Conjugates (Immunoconjugates)

Anti-Ovr110 antibody-maytansinoid conjugates are prepared by chemicallylinking an anti-Ovr110 antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al. Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred. Conjugates of the antibody and maytansinoid maybe made using a variety of bifunctional protein coupling agents such asN-succinimidyl (2-pyridyldithio) propionate (SPDP),succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl (2-pyridyldithio) propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) and N-succinimidyl(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. Preferably, the linkage is formedat the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-Ovr110 antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁ ^(I), (Hinman etal. Cancer Research 53: 3336 (1993), Lode et al. Cancer Research 5 8:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-Ovr110antibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain, 15nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, WO 93/21232 published Oct. 28, 1993. The presentinvention further contemplates an immunoconjugate formed between anantibody and a compound with nucleolytic activity (e.g. a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-Ovr110 antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²,and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example Tc^(99M) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99M), I¹²³, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such as N-succinimidyl(2-pyridyldithio) propionate (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT),bifunctional derivatives of imidoesters (such as dimethyl adipimidateHCL), active esters (such as disuccinimidyl suberate), aldehydes (suchas glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-Ovr110 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

In addition, the antibody may be conjugated to a “receptor” (suchstreptavidin) for utilization in tumor pre-targeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asO-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; P-lactamase useful for converting drugsderivatized with P-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population. The enzymes of this invention can becovalently bound to the anti-Ovr110 antibodies by techniques well knownin the art such as the use of the heterobifunctional crosslinkingreagents discussed above.

Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984).

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-Ovr110 antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acid molecule encoding thehumanized anti-Ovr110 antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody. For recombinant production of the antibody, the nucleic acidmolecule encoding it is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or inserted into a vectorin operable linkage with a promoter for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to nucleic acid molecules encoding the heavy andlight chains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

The anti-Ovr110 antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native anti-Ovr110 antibody signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, oc factor leader (including Saccharomyces andKluyveromyces cc-factor leaders), or acid phosphatase leader, the Calbicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor region is ligated in reading frameto DNA encoding the anti-Ovr110 antibody.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-Ovr110 antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -11, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. For example, cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-Ovr110 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4 Jones, Genetics, 85:12 (1977). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDI canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theanti-Ovr110 antibody nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, P-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgamo (S.D.) sequenceoperably linked to the DNA encoding the anti-Ovr110 antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors. Examples of suitable promoter sequences for use withyeast hosts include the promoters for 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-Ovr110 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human P-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding the anti-Ovr110 antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theanti-Ovr110 antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-Ovr110 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-Ovr110antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-Ovr110antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be utilized as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-Ovr110 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Culturing Host Cells

The host cells used to produce the anti-Ovr110 antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's FIO (Sigma), Minimal Essential Medium (MEM)(Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM)(Sigma)are suitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Anti-Ovr110 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of Ecoli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SIDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Pharmaceutical formulations of the antibodies used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate, Tris,phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol, and mcresol); low molecular weight(less than about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyllolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theantibody preferably comprises the antibody at a concentration of between5-200 mg/ml, preferably between 10-100 mg/ml.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, in addition to the anti-Ovr110 antibody which internalizes,it may be desirable to include in the one formulation, an additionalantibody, e.g. a second anti-Ovr110 antibody which binds a differentepitope on Ovr110, or an antibody to some other target such as a growthfactor that affects the growth of the particular cancer. Alternatively,or additionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively,in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−) hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Methods and Treatment Using Anti-Ovr110 Antibodies

According to the present invention, the anti-Ovr110 antibody thatinternalizes upon binding Ovr110 on a cell surface is used to treat asubject in need thereof having a cancer characterized byOvr110-expressing cancer cells, in particular, head and neck, ovarian,pancreatic, lung, endometrial or breast cancer, more particularly, headand neck adenoid cystic carcinoma, ovarian serous adenocarcinoma orbreast infiltrating ductal carcinoma cancer, and associated metastases.

The cancer will generally comprise Ovr110-expressing cells, such thatthe anti-Ovr110 antibody is able to bind thereto. While the cancer maybe characterized by overexpression of the Ovr110 molecule, the presentapplication further provides a method for treating cancer which is notconsidered to be an Ovr110-overexpressing cancer.

This invention also relates to methods for detecting cells whichoverexpress Ovr110 and to diagnostic kits useful in detecting cellsexpressing Ovr110 or in detecting Ovr110 in serum from a patient. Themethods may comprise combining a cell-containing test sample with anantibody of this invention, assaying the test sample for antibodybinding to cells in the test sample and comparing the level of antibodybinding in the test sample to the level of antibody binding in a controlsample of cells. A suitable control is, e.g., a sample of normal cellsof the same type as the test sample or a cell sample known to be free ofOvr110 overexpressing cells. A level of Ovr110 binding higher than thatof such a control sample would be indicative of the test samplecontaining cells that overexpress Ovr110. Alternatively the control maybe a sample of cells known to contain cells that overexpress Ovr110. Insuch a case, a level of Ovr110 antibody binding in the test sample thatis similar to, or in excess of, that of the control sample would beindicative of the test sample containing cells that overexpress Ovr110.

Ovr110 overexpression may be detected with a various diagnostic assays.For example, over expression of Ovr110 may be assayed byimmunohistochemistry (IHC). Parrafin embedded tissue sections from atumor biopsy may be subjected to the IHC assay and accorded an Ovr110protein staining intensity criteria as follows.

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for Ovr110 expression may becharacterized as not overexpressing Ovr110, whereas those tumors with 2+or 3+ scores may be characterized as overexpressing Ovr110.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Arizona) or PATHVISION™ (VySiS, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of Ovr110 overexpression in the tumor. Ovr110 overexpression oramplification may be evaluated using an in vivo diagnostic assay, e.g.by administering a molecule (such as an antibody of this invention)which binds Ovr110 and which is labeled with a detectable label (e.g. aradioactive isotope or a fluorescent label) and externally scanning thepatient for localization of the label.

A sample suspected of containing cells expressing or overexpressingOvr110 is combined with the antibodies of this invention underconditions suitable for the specific binding of the antibodies toOvr110. Binding and/or internalizing the Ovr110 antibodies of thisinvention is indicative of the cells expressing Ovr110. The level ofbinding may be determined and compared to a suitable control, wherein anelevated level of bound Ovr110 as compared to the control is indicativeof Ovr110 overexpression. The sample suspected of containing cellsoverexpressing Ovr110 may be a cancer cell sample, particularly a sampleof an ovarian cancer, e.g. ovarian serous adenocarcinoma, a breastcancer, e.g., a breast infiltrating ductal carcinoma, lung cancer,pancreatic cancer, head and neck cancer, e.g. adenoid cystic carcinoma,or endometrial cancer. A serum sample from a subject may also be assayedfor levels of Ovr110 by combining a serum sample from a subject with anOvr110 antibody of this invention, determining the level of Ovr110 boundto the antibody and comparing the level to a control, wherein anelevated level of Ovr110 in the serum of the patient as compared to acontrol is indicative of overexpression of Ovr110 by cells in thepatient. The subject may have a cancer such as e.g., an ovarian cancer,e.g. ovarian serous adenocarcinoma, a breast cancer, e.g., a breastinfiltrating ductal carcinoma, lung cancer, pancreatic cancer, head andneck cancer, e.g. adenoid cystic carcinoma, or endometrial cancer.

Currently, depending on the stage of the cancer, hand and neck, ovarian,pancreatic, lung, endometrial or breast cancer treatment involves one ora combination of the following therapies: surgery to remove thecancerous tissue, radiation therapy, androgen deprivation (e.g.,hormonal therapy), and chemotherapy. Anti-Ovr110 antibody therapy may beespecially desirable in elderly patients who do not tolerate thetoxicity and side effects of chemotherapy well, in metastatic diseasewhere radiation therapy has limited usefulness, and for the managementof prostatic carcinoma that is resistant to androgen deprivationtreatment. The tumor targeting and internalizing anti-Ovr110 antibodiesof the invention are useful to alleviate Ovr110-expressing cancers,e.g., ovarian, pancreatic, head and neck, lung, endometrial or breastcancers upon initial diagnosis of the disease or during relapse. Fortherapeutic applications, the anti-Ovr110 antibody can be used alone, orin combination therapy with, e.g., hormones, antiangiogens, orradiolabelled compounds, or with surgery, cryotherapy, and/orradiotherapy, notably for ovarian, pancreatic, head and neck, lung,endometrial or breast cancers, also particularly where shed cells cannotbe reached. Anti-Ovr110 antibody treatment can be administered inconjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy, Chemotherapeuticdrugs such as Taxotere® (docetaxel), Taxol® (paclitaxel), estramustineand mitoxantrone are used in treating metastatic and hormone refractoryovarian, pancreatic, lung or breast cancer, in particular, in good riskpatients. In the present method of the invention for treating oralleviating cancer, in particular, androgen independent and/ormetastatic ovarian, pancreatic, lung, endometrial, head and neck orbreast cancer, the cancer patient can be administered anti-Ovr110antibody in conjunction with treatment with the one or more of thepreceding chemotherapeutic agents. In particular, combination therapywith paclitaxel and modified derivatives (see, e.g., EP0600517) iscontemplated. The anti-Ovr110 antibody will be administered with atherapeutically effective dose of the chemotherapeutic agent. Theanti-Ovr110 antibody may also be administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

Particularly, an immunoconjugate comprising the anti-Ovr110 antibodyconjugated with a cytotoxic agent may be administered to the patient.Preferably, the immunoconjugate bound to the Ovr110 protein isinternalized by the cell, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the cancer cell to which it binds.Preferably, the cytotoxic agent targets or interferes with the nucleicacid in the cancer cell. Examples of such cytotoxic agents are describedabove and include maytansin, maytansinoids, saporin, gelonin, ricin,calicheamicin, ribonucleases and DNA endonucleases.

The anti-Ovr110 antibodies or immunoconjugates are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies or immunoconjugates may beinjected directly into the tumor mass. Intravenous or subcutaneousadministration of the antibody is preferred. Other therapeutic regimensmay be combined with the administration of the anti-Ovr110 antibody.

The combined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Preferably such combined therapy results in asynergistic therapeutic effect.

It may also be desirable to combine administration of the anti-Ovr110antibody or antibodies, with administration of an antibody directedagainst another tumor antigen associated with the particular cancer. Assuch, this invention is also directed to an antibody “cocktail”comprising one or more antibodies of this invention and at least oneother antibody which binds another tumor antigen associated with theOvr110-expressing tumor cells. The cocktail may also comprise antibodiesthat are directed to other epitopes of Ovr110. Preferably the otherantibodies do not interfere with the binding and or internalization ofthe antibodies of this invention.

The antibody therapeutic treatment method of the present invention mayinvolve the combined administration of an anti-Ovr110 antibody (orantibodies) and one or more chemotherapeutic agents or growth inhibitoryagents, including co-administration of cocktails of differentchemotherapeutic agents. Chemotherapeutic agents include, e.g.,estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil,melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such aspaclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams and Wilkins, Baltimore, Md. (1992).

The antibody may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (see, EP 616 812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated isandrogen independent cancer, the patient may previously have beensubjected to anti-androgen therapy and, after the cancer becomesandrogen independent, the anti-Ovr110 antibody (and optionally otheragents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy, before, simultaneouslywith, or post antibody therapy. Suitable dosages for any of the aboveco-administered agents are those presently used and may be lowered dueto the combined action (synergy) of the agent and anti-Ovr110 antibody.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Preferably, the antibody isadministered by intravenous infusion or by subcutaneous injections.Depending on the type and severity of the disease, about 1 pg/kg toabout 50 mg/kg body weight (e.g. about 0.1-15 mg/kg/dose) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A dosing regimen can comprise administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the anti-Ovr110 antibody. However, other dosageregimens may be useful. A typical daily dosage might range from about 1pg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of this therapy can be readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of a nucleic acid molecule encoding theantibody is encompassed by the expression “administering atherapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy togenerate intracellular antibodies.

There are two major approaches to introducing the nucleic acid molecule(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid molecule is injecteddirectly into the patient, usually at the site where the antibody isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid molecule is introduced into these isolated cells and themodified cells are administered to the patient either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). Thereare a variety of techniques available for introducing nucleic acidmolecules into viable cells. The techniques vary depending upon whetherthe nucleic acid is transferred into cultured cells in vitro, or in vivoin the cells of the intended host. Techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,the calcium phosphate precipitation method, etc. A commonly used vectorfor ex vivo delivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid molecule transfertechniques include transfection with viral vectors (such as adenovirus,Herpes simplex I virus, or adeno-associated virus) and lipid-basedsystems (useful lipids for lipid-mediated transfer of the gene areDOTMA, DOPE and DC-Chol, for example). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Articles of Manufacture and Kits

The invention also relates to an article of manufacture containingmaterials useful for the detection for Ovr110 overexpressing cellsand/or the treatment of Ovr110 expressing cancer, in particular ovarian,pancreatic, head and neck, lung, endometrial or breast cancer. Thearticle of manufacture comprises a container and a composition containedtherein comprising an antibody of this invention. The composition mayfurther comprise a carrier. The article of manufacture may also comprisea label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fordetecting Ovr110 expressing cells and/or treating a cancer condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an anti-Ovr110 antibody of the invention. The label orpackage insert indicates that the composition is used for detectingOvr110 expressing cells and/or for treating ovarian, pancreatic, lung,head and neck, endometrial or breast cancer, or more specifically headand neck adenoid cystic carcinoma, ovarian serous adenocarcinoma orbreast infiltrating ductal carcinoma cancer, in a patient in needthereof. The label or package insert may further comprise instructionsfor administering the antibody composition to a cancer patient.Additionally, the article of manufacture may further comprise a secondcontainer comprising a substance which detects the antibody of thisinvention, e.g., a second antibody which binds to the antibodies of thisinvention. The substance may be labeled with a detectable label such asthose disclosed herein. The second container may contain e.g., apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forOvr110 cell killing assays, for purification or immunoprecipitation ofOvr110 from cells or for detecting the presence of Ovr110 in a serumsample or detecting the presence of Ovr110-expressing cells in a cellsample. For isolation and purification of Ovr110, the kit can contain ananti-Ovr110 antibody coupled to a solid support, e.g., a tissue cultureplate or beads (e.g., sepharose beads). Kits can be provided whichcontain the antibodies for detection and quantitation of Ovr110 invitro, e.g. in an ELISA or a Western blot. As with the article ofmanufacture, the kit comprises a container and a composition containedtherein comprising an antibody of this invention. The kit may furthercomprise a label or package insert on or associated with the container.The kits may comprise additional components, e.g., diluents and buffers,substances which bind to the antibodies of this invention, e.g., asecond antibody which may comprise a label such as those disclosedherein, e.g., a radiolabel, fluorescent label, or enzyme, or the kit mayalso comprise control antibodies. The additional components may bewithin separate containers within the kit. The label or package insertmay provide a description of the composition as well as instructions forthe intended in vitro or diagnostic use.

EXAMPLES Example 1 Monoclonal Antibody Producing Hybridomas

The following MAb/hybridomas of the present invention are describedbelow:

Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1,Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1,Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107,Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3,Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1,Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15,Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2,Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9,Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.I5, Ovr110.I16,Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1,Ovr110.J2 and Ovr110.J3. If the MAb has been cloned, it will get thenomenclature “X.1,” e.g., the first clone of A7 will be referred to asA7.1, the second clone of A7 will be referred to as A7.2, etc. For thepurposes of this invention, a reference to A7 will include all clones,e.g., A7.1, A7.2, etc.

These monoclonal antibodies (including physical, functional andbiochemical properties) and the hybridomas producing these antibodieshave been previously described in PCT/US2004/014490 andPCT/US2005/040707, the contents of which are herein incorporated byreference.

Example 2 IHC of Ovr110 Expression in Head and Neck and EndometrialCancers

Head and neck adenoid cystic carcinoma (ACC) and endometrial cancersamples from various tumor locations were evaluated for Ovr110expression by Immunohistochemistry (IHC).

Formalin-fixed, paraffin-embedded tissue blocks of normal or canceroustissues were retrieved from the Surgical Pathology collections of theUniversity of Colorado Health Science Center (UCHSC). Tissues weresectioned to 5 μm, mounted on charged glass slides (Superfrost Plus;Fisher Scientific, Pittsburgh, Pa.), and baked overnight at 60° C.Deparaffinization with xylene and rehydration through a graded alcoholseries was followed by blocking of the endogenous peroxidase activitywith 3.0% hydrogen peroxide for 15 minutes. Antigen retrieval wasperformed in a decloaking chamber (Biocare Medical, Walnut Creek,Calif.) by heating the slides in citrate buffer (10-20 mmol/l, pH 6.0)at 120° C. for 10 minutes. Immunohistochemical staining for Ovr110 wasperformed using mouse monoclonal antibody Ovr110.A57.1 (ATCC depositnumber PTA-5180) at a final working concentration of 0.4 ug/ml.

Negative controls for the mouse antibodies were performed on allsections using an equivalent concentration of a subclass-matched IgG1 K(Becton Dickson PharMingen), that was generated against unrelatedantigens. Antigen expression was visualized by development with3,3′-diaminobenzidine (DakoCytomation) and the sections werecounterstained with hematoxylin, dehydrated in graded alcohols, andfinally coverslipped.

All slides were reviewed by Board Certified Anatomic and ClinicalPathologists at the UCHSC. Ovr110 staining intensity of the tumor cellswas graded on a scale of 1 to 4+ where 1+ staining reflected faintlyperceptible positive results and 4+ staining corresponded to levelsequivalent to that seen in a previously characterized strong positivecontrol section (ovarian serous carcinoma). The percentage of tumorcells that stained for Ovr110 was evaluated by review of the completehistologic section from each case (>0-100%).

Ovr110 Expression in Adenoid Cystic Carcinoma

Twenty-three archival formalin-fixed, paraffin-embedded tissue blocks ofhead and neck adenoid cystic carcinoma (ACC) were evaluated for Ovr110as described above. Detectable Ovr110 expression was observed in 23/23(100%) invasive adenoid cystic carcinomas samples and intense (3+ to 4+)cytoplasmic and cell surface Ovr110 staining was detected in >90% oftumor cells in 20/23 samples. See Table 1 below. Ovr110 staining clearlydelineated the extent of the tumor, including foci of perineuralinvasion. See FIGS. 1B and 1C. Negative control sections showed noevidence of non-specific staining and preincubation of the Ovr110.A57.1antibody with the full-length recombinant Ovr110 protein completelyblocked the staining in histologic sections (FIG. 1A).

TABLE 1 Ovr110 staining in ACC Ovr110 Proportion of Sample DiseaseStaining Cells Positive ID Diagnosis Tumor Location Intensity for Ovr110A5 ACC Tongue 3+ 100% A6 ACC Parotid 3+ 100% A7 ACC nose/frontal 3+ 100%A8 ACC Palate 3+ 98% A10 ACC skin forehead 4+ 100% A11 ACC right neck 3+100% A12 ACC submandibular gland 3+ 90% A13 ACC Glottis 3+ 100% A14 ACCethmoid sinus 4+ 100% A15 ACC Epiglottis 3+ 90% A16 ACC Palate 4+ 100%A17 ACC right buccal space 3+ 100% A18 ACC parotid gland + nerve 1+ 30%A19 ACC parotid gland 3+ 100% A20 ACC submandibular gland 4+ 100% A21ACC parotid gland 4+ 100% A22 ACC facial nerve 4+ 100% A23 ACC Larynx 4+100% A24 ACC neck mass biopsy 3+ 100% A25 ACC cheek mass 3+ 2% A26 ACCsubmandibular gland 2+ 80% A27 ACC neck mass 3+ 100% A28 ACCsubmandibular gland 4+ 100%

Ovr110 Expression in Uterine Endometrial Cancer

Ninety-one archival formalin-fixed, paraffin-embedded tissue blocks ofuterine endometrial adenocarcinoma were evaluated for Ovr110 asdescribed above. Slides were reviewed as above except Ovr110 stainingintensity of the tumor cells was graded on a scale of 1 to 3+ where 1+staining reflected faintly perceptible positive results and 3+ stainingcorresponded to strong positive staining. The percentage of tumor cellsthat stained for Ovr110 was evaluated by review of the completehistologic section from each case (>0-100%).

Detectable Ovr110 expression was observed in 91/91 (100%) invasiveuterine endometrial adenocarcinoma samples and intense (2+ to 3+)cytoplasmic and cell surface Ovr110 staining was detected in 88/91samples. The percent of Ovr110 positive cells and Ovr110 stainingintensity was compared to tumor stage and grade as well as the age ofthe individual at time of diagnosis and are summarized Tables 2 and 3below.

TABLE 2 Proportion of Ovr110 Positive Cells Compared toClinical-Pathologic Variables No. Cases (%) grouped by proportion ofOvr110-positive cells No. Cases 0-10% >10-50% >50-80% >80-100% 1 48 0(0%) 11 (23%) 17 (35%) 20 (42%) Grade 2 30 2 (7%) 7 (23%) 5 (17%) 16(53%) 3 13 2 (15%) 2 (15%) 3 (23%) 6 (46%) I 65 1 (2%) 16 (25%) 17 (26%)31 (48%) Stage II 7 0 (0%) 1 (14%) 2 (29%) 4 (57%) III-IV 19 3 (16%) 3(16%) 6 (32%) 7 (37%) Age at ≦50 23 2 (9%) 9 (39%) 7 (30%) 5 (22%)diagnosis >50 68 2 (3%) 11 (16%) 18 (26%) 37 (54%) Due to rounding,percentages in parentheses may not add up to 100%

TABLE 3 Median Ovr110 Staining Intensity Compared to Clinical-PathologicVariables Ovr110 Staining intensity No. Cases 1+ 2+ 3+ 1 48 2 (4%) 19(40%) 27 (56%) Grade 2 30 1 (3%) 13 (43%) 16 (53%) 3 13 0 (0%) 3 (23%)10 (77%) I 65 3 (5%) 26 (40%) 36 (55%) Stage II 7 0 (0%) 3 (43%) 4 (57%)III-IV 19 0 (0%) 6 (32%) 13 (68%) Age at ≦50 23 2 (9%) 13 (57%) 8 (35%)diagnosis >50 68 1 (1%) 22 (32%) 45 (66%) Due to rounding, percentagesin parentheses may not add up to 100%

Additionally, the uterine endometrial adenocarcinoma samples wereevaluated by carcinoma type compared to Ovr110 positive cells and Ovr110staining intensity. Type I carcinomas were defined as grade 1, stage Iand individuals diagnosed at under 50 years old. Type II carcinomas weredefined as all other uterine endometrial cancer cases (grade 2, 3 andstage II, III, IV) who were diagnosed at over 50 years old. Atypicalcases were eliminated and 11 cases of type I and 68 cases of type IIuterine endometrial cancer were evaluated. The results are summarized intables 4 and 5 below.

TABLE 4 Proportion of Ovr110 Positive Cells Compared to Tumor Type No.Cases (%) grouped by proportion of No. Ovr110-positive cells Cases0-10% >10-50% >50-80% >80-100% p-value* Tumor I 11 0 (0%) 6 (55%) 3(27%) 2 (18%) 0.032 type II 68 2 (3%) 11 (16%) 18 (27%) 37 (54%) Due torounding, percentages in parentheses may not add up to 100%. *Fisher'sexact test for tables larger than 2 × 2 tables

TABLE 5 Proportion of Ovr110 Positive Cells Compared to Tumor TypeOvr110 Staining intensity No. p- Cases 1+ 2+ 3+ value* Tumor I 11 2(18%) 7 (64%) 2 (18%) 0.003 type II 68 1 (1%) 22 (32%) 45 (66%) Due torounding, percentages in parentheses may not add up to 100%. *Fisher'sexact test for tables larger than 2 × 2 tablesThese results demonstrate that Ovr110 is highly expressed in uterineendometrial adenocarcinoma. Furthermore, high expression of Ovr110 iscorrelated with more aggressive tumors (grade 2, 3 and stage II, III,IV) in individuals over 50 years old.

Summary of Ovr110 Expression in Head and Neck and Uterine EndometrialCancer

These results demonstrate that Ovr110 is highly expressed in adenoidcystic carcinoma (ACC) and uterine endometrial adenocarcinoma. We havepreviously shown that Ovr110 is expressed in low levels in normal andnon-cancerous tissues. See PCT/US2004/014490 and PCT/US2005/040707.Thus, blocking or inhibiting Ovr110 over-expression or Ovr110 activity(including binding to a receptor) is useful for killing cancer cells,preventing growth of cancer cells, preventing growth/metastases oftumors and shrinking tumors. Anti-Ovr110 compounds such as theantibodies described above bind to cancer cells over-expressing Ovr110and inhibit Ovr110 function resulting in therapeutic benefits againstcancer cells and tumors.

Example 3 Inverse Correlation of T Cell Invasion to Ovr110 Expression inTumors

Tumor samples from various tissues were evaluated byimmunohistochemistry (IHC) for Ovr110 expression and tumor-associated Tcell lymphocyte infiltration. T cell lymphocyte infiltration wasevaluated by staining for CD3, CD8 and/or CD69. Positive CD3 stainingindicates the presence of peripheral T cell lymphocytes and positive CD8staining indicates the presence of suppressor/cytotoxic T celllymphocytes which promote immune response to tumors. CD69 antigen is notexpressed on resting peripheral blood lymphocytes but is amongst theearliest antigens to appear upon activation of lymphocytes. PositiveCD69 staining indicates the presence of activated suppressor/cytotoxic Tcell lymphocytes.

IHC Methods

Formalin-fixed, paraffin-embedded tissue blocks of normal or canceroustissues were retrieved from the Surgical Pathology collections of theUniversity of Colorado Health Science Center (UCHSC) forimmunohistochemical (IHC) evaluation. Tissues were sectioned to 5 μm,mounted on charged glass slides (Superfrost Plus; Fisher Scientific,Pittsburgh, Pa.), and baked overnight at 60° C. Deparaffinization withxylene and rehydration through a graded alcohol series was followed byblocking of the endogenous peroxidase activity with 3.0% hydrogenperoxide for 15 minutes. Antigen retrieval was performed in a decloakingchamber (Biocare Medical, Walnut Creek, Calif.) by heating the slides incitrate buffer (10-20 mmol/l, pH 6.0) at 120° C. for 10 minutes.Immunohistochemical staining for Ovr110 was performed using mousemonoclonal antibody Ovr1110.A57.1 (ATCC deposit number PTA-5180) at afinal working concentration of 0.4 ug/ml. Immunohistochemical stainingfor CD3 and CD8 was performed using DakoCytomation antibodies, CD3(rabbit polyclonal, ref.# A0452) and CD8 (mouse monoclonal, ref.#M7103)at respective final working concentrations of 2 ug/ml and 0.25 ug/ml.Immunohistochemical staining for CD69 was performed using mousemonoclonal antibody CD69 Ab-1 (Cat. #MS-1478) from Lab VisionCorporation (Fremont, Calif.) at a dilution of 1:20 from manufacturesstock. Immunohistochemical staining for cleaved Caspase-3 was performedusing rabbit polyclonal antibody Asp 175 (Cell Signaling Inc., Danvers,Mass.) at a final working concentration of 0.6 ug/ml. Tissue stainingfor Ovr110, CD3 and cleaved Caspase-3 were performed on an Autostainer(DakoCytomation, Carpentaria, Calif.) by an indirect avidin-biotin basedimmunoperoxidase method (Vector Laboratories, Burlingame, Calif.). CD8staining was performed on the Autostainer using the Envisionimmunoperoxidase detection system (DakoCytomation).

Negative controls for the mouse antibodies were performed on allsections using an equivalent concentration of a subclass-matched IgG1 K(Becton Dickson PharMingen), that was generated against unrelatedantigens. Negative controls for the rabbit antibodies were performedusing an equivalent concentration of rabbit polyclonal immunoglobulin(DakoCytomation). Antigen expression was visualized by development with3,3′-diaminobenzidine (DakoCytomation) and the sections werecounterstained with hematoxylin, dehydrated in graded alcohols, andfinally coverslipped.

All slides were reviewed by Board Certified Anatomic and ClinicalPathologists at the UCHSC. Ovr110 staining intensity of the tumor cellswas graded on a scale of 1 to 4+ where 1+ staining reflected faintlyperceptible positive results and 4+ staining corresponded to levelsequivalent to that seen in a previously characterized strong positivecontrol section (ovarian serous carcinoma). The percentage of tumorcells that stained for Ovr110 was evaluated by review of the completehistologic section from each case (>0-100%).

CD3, CD8 and CD69 scores were evaluated based on the staining ofperi-tumoral and infiltrative lymphocytes as negative, 1+, 2+ or 3+,corresponding respectively with the detection of <1, 1-20, 20-100, andgreater than 100 stained lymphocytes per 200× microscopic field, asaveraged over the entire tumor-involved section. The CD3, CD8 and CD69scores were correlated with the results of Ovr110 immunohistochemicallocalization, number of Ovr110 positive cells and/or Ovr110 stainingintensity by comparison of results of matched serial sections from eachcase.

The Apoptosis Index (A.I.) was calculated from the cleaved Caspase-3staining results. A.I. is the proportion of cells positive for cleavedCaspase-3 within tumor cells.

IHC Results for Adenoid Cystic Carcinoma

Twenty-three archival formalin-fixed, paraffin-embedded tissue blocks ofhead and neck adenoid cystic carcinoma (ACC) were evaluated for Ovr110,CD3 and CD8 expression as described above.

As reported above, Ovr110 expression was observed in 23/23 (100%)invasive adenoid cystic carcinomas samples and intense (3+ to 4+)cytoplasmic and cell surface Ovr110 staining was detected in >90% oftumor cells in 20/23 samples.

CD3 and CD8 immunohistochemical stains detected both peritumoral andtumor infiltrative lymphocytes. The CD3 positive lymphocytes scoresranged from 0 to 3+(range 0 to 3+, mean 0.95, Std. Dev. 1.17),corresponding to zero to greater than 100 lymphocytes/200× microscopicfield. See FIG. 2B. In general, the tumors showed lower CD8 scores(range 0 to 3+, mean 0.39, Std. Dev. 0.72). See FIG. 3B. Table 6 belowsummarizes the results of IHC staining.

TABLE 6 Ovr110, CD3 positive T cells and CD8 positive T cells stainingin ACC Proportion Disease Ovr110 of Cells CD8 Sample Diagno- StainingPositive CD3 Staining Staining ID sis Intensity for Ovr110 IntensityIntensity A5 ACC 3+ 100% 1+ 1+ focal A6 ACC 3+ 100% neg neg A7 ACC 3+100% neg neg A8 ACC 3+  98% 1+ neg A10 ACC 4+ 100% neg neg A11 ACC 3+100% 1+ neg A12 ACC 3+  90% 3+ 1+ A13 ACC 3+ 100% neg neg A14 ACC 4+100% focal 3+ neg A15 ACC 3+  90% neg 1+ A16 ACC 4+ 100% focal 3+, negmostly neg A17 ACC 3+ 100% 2+ 2+ A18 ACC 1+  30% focal 3+ 2+ A19 ACC 3+100% neg neg A20 ACC 4+ 100% 1+ neg A21 ACC 4+ 100% neg neg A22 ACC 4+100% 1+ neg A23 ACC 4+ 100% neg neg A24 ACC 3+ 100% neg neg A25 ACC 3+ 2% neg neg A26 ACC 2+  80% 2+ 2+ A27 ACC 3+ 100% neg neg A28 ACC 4+100% neg neg

Comparison of histochemical stains in matched serial sections revealedan inverse correlation between intensity and percent positive of Ovr110expressing tumor cells and tumor-associated T cells. Tumors with higherlevels of Ovr110 expression (16/20) demonstrated fewer (0 to 1+) CD3lymphocytes. Similarly, 19/20 cases of ACC with higher Ovr110 scores hadfewer CD8 positive T cells. By contrast, ⅔ ACC samples with lowerpercentages of Ovr110 positive cells had increased CD3 and CD8lymphocyte scores.

IHC Results for Endometrial Cancer

Ninety-one archival formalin-fixed, paraffin-embedded tissue blocks ofuterine endometrial adenocarcinoma were evaluated for Ovr110, CD3, CD8and cleaved Capase-3 expression as described above. Slides were reviewedas above except Ovr110 staining intensity of the tumor cells was gradedon a scale of 1 to 3+ where 1+ staining reflected faintly perceptiblepositive results and 3+ staining corresponded to strong positivestaining. The percentage of tumor cells that stained for Ovr110 wasevaluated by review of the complete histologic section from each case(>0-100%).

As reported above, Ovr110 expression was observed in 91/91 (100%)invasive uterine endometrial adenocarcinoma samples and intense (2+ to3+) cytoplasmic and cell surface Ovr110 staining was detected in 88/91samples.

CD3 and CD8 immunohistochemical stains detected lymphocytes at the tumorborder and tumor infiltrating lymphocytes. The number of CD3 and CD8positive cells per 10 high power fields. Table 7 below summarizes theresults of IHC staining showing the median number of CD3 and CD8positive cells infiltrating the tumor and at the tumor border with therange in parenthesis. Also listed is the Apoptosis Index (A.I.).

TABLE 7 Ovr110, CD3 positive T cells and CD8 positive T cells stainingin endometrial cancer CD8 CD3 score CD3 score score at CD8 score attumor infiltrat tumor infiltrat Cases border tumor border tumor A.I. (%)Ovr110  0-10% 4 1169  26 171  6 0.98 Positive (318-1732) (7-83) (98-972)  (4-37)  (0.39-1.12) cells >10-50% 20 767 71 191 36 0.26(125-3230) (4-1148) (6-1214) (1-377) (0.00-1.67) >50-80% 25 473 40  79 9 0.18  (36-4290) (6-374)  (4-732)  (0-95)  (0.00-0.89)  >80-100% 42558 49  74 11 0.30  (34-3621) (1-1095) (3-1473) (0-220) (0.00-5.54)These results demonstrate that the median number of peripheral T celllymphocytes (CD3 positive) and suppressor/cytotoxic T cell lymphocytes(CD8 positive) is significantly decreased overall in tumors with >50%Ovr110 positive cells (1120 CD3+, 173 CD8+) compared to those with ≦50%Ovr110 positive cells (2703 CD3+, 404 CD8+). Also evident in tumorswith >50% Ovr110 positive cells is that the median number ofinfiltrating CD3 positive and CD8 positive lymphocytes (89 CD3+, 20CD8+) is lower compared to peritumoral CD3 positive and CD8 positivelymphocytes (1031 CD3+, 153 CD8+). The apoptosis index is also loweroverall in tumors with >50% Ovr110 positive cells than in tumors with≦50% Ovr110 positive cells.

These results demonstrate that Ovr110 plays a role in shielding tumorsfrom immune cell surveillance, limiting T cell tumor infiltration andreducing apoptosis of tumor cells.

IHC Results for Breast Cancer

Archival formalin-fixed, paraffin-embedded tissue blocks of invasivemalignant breast cancer and invasive ductal breast carcinomas werestained for Ovr110, CD3 and CD8 expression as described above. Breastcancer samples were evaluated for proportion (percentage) of cancercells positive for Ovr110 expression, Ovr110 expression pattern andOvr110 staining intensity. Ovr110 expression patterns were categorizedas Pattern 1: apical membranous staining; Pattern 2: mixture of pattern1 and pattern 3; and Pattern 3: circumferential and cytoplasmicstaining. Ovr110 staining intensity of the tumor cells was graded on ascale of 1 to 3+ where 1+ staining reflected faintly perceptiblepositive results and 3+ staining corresponded to strong positivestaining. Lymphocyte staining was categorized into four groups based onaverage number of lymphocytes per high power field (hpf). Group 0: ≦5cells/hpf; Group 1: 6-50 cells/hpf; Group 2: 51-99 cells/hpf; and Group3: ≧100 cells/hpf. Groups 0 and 1 were considered low lymphocyte countsand Groups 2 and 3 were considered high lymphocyte counts in the resultsbelow.

CD3 and CD8 immunohistochemical stains detected tumor infiltratinglymphocytes in breast cancer samples. Tables 8-11 below summarize theresults of IHC staining showing the median proportion of Ovr110 positivecells (in number of samples), the median Ovr110 staining pattern (innumber of samples) and median Ovr110 staining intensity (in number ofsamples) per low lymphocyte invasion (Groups 0 and 1) and highlymphocyte invasion (Groups 2 and 3).

TABLE 8 Ovr100 infiltrating CD3 T cells staining in invasive malignantbreast cancer Infiltrating CD3 positive T cells Low High Group† Group‡Median proportion Ovr110 positive cells (# of 99 (18) 80 (17) cases)Median Ovr110 staining pattern (# of cases) 3 (18) 3 (15) Median Ovr110staining intensity (# of cases) 3 (18) 3 (15) †Low = Group 0 (≦5 lymphsper 3 hpf) and Group 1 (6-50 lymphs per 3 hpf) ‡High = Group 2 (51-99lymphs per 3 hpf) and Group 3 (≧100 per 3 hpf)

TABLE 9 Ovr110 infiltrating CD8 T cells staining in invasive malignantbreast cancer Infiltrating CD8 positive T cells Low High Group† Group‡Median proportion Ovr110 positive cells (# of 98 (19) 70 (16) cases)Median Ovr110 staining pattern (# of cases) 3 (19) 3 (14) Median Ovr110staining intensity (# of cases) 3 (19) 2 (14) †Low = Group 0 (≦5 lymphsper 3 hpf) and Group 1 (6-50 lymphs per 3 hpf) ‡High = Group 2 (51-99lymphs per 3 hpf) and Group 3 (≧100 per 3 hpf)

TABLE 10 Ovr110 infiltrating CD3 T cells staining in invasive ductalbreast cancer Infiltrating CD3 positive T cells Low High Group† Group‡Median proportion Ovr110 positive cells (# of 100 (11) 30 (8) cases)Median Ovr110 staining pattern (# of cases) 3 (11) 3 (6) Median Ovr110staining intensity (# of cases) 3 (11) 2 (6) †Low = Group 0 (≦5 lymphsper 3 hpf) and Group 1 (6-50 lymphs per 3 hpf) ‡High = Group 2 (51-99lymphs per 3 hpf) and Group 3 (≧100 per 3 hpf)

TABLE 11 Ovr110 infiltrating CD8 T cells staining in invasive ductalbreast cancer Infiltrating CD8 positive T cells Low High Group† Group‡Median proportion Ovr110 positive cells (# of 100 (11) 30 (8) cases)Median Ovr110 staining pattern (# of cases) 3 (11) 3 (6) Median Ovr110staining intensity (# of cases) 3 (11) 2 (6) †Low = Group 0 (≦5 lymphsper 3 hpf) and Group 1 (6-50 lymphs per 3 hpf) ‡High = Group 2 (51-99lymphs per 3 hpf) and Group 3 (≧100 per 3 hpf)

These results demonstrate the number of peripheral T cell lymphocytes(CD3 positive) and suppressor/cytotoxic T cell lymphocytes (CD8positive) is reduced in breast cancers that highly express Ovr110.Additionally, fewer cases with high levels of infiltrating lymphocytes(High group) were observed in when Ovr110 staining intensity was high(3+). These results demonstrate that Ovr110 plays a role in limiting Tcell tumor infiltration.

IHC Results for Ovarian Cancer

Archival formalin-fixed, paraffin-embedded tissue blocks of ovariancancer are evaluated for Ovr110, CD3 and CD8 expression as describedabove. CD3, CD8 and CD69 immunohistochemical stains detect lymphocytesat the tumor border and tumor infiltrating lymphocytes. The number ofCD3, CD8 and CD69 positive cells per 10 high power fields. Results ofIHC staining show the median number of CD3, CD8, CD69 positive cellsinfiltrating the tumor and at the tumor border with the range inparenthesis.

In ovarian cancer the median number of peripheral and invasive T celllymphocytes (CD3 positive), suppressor/cytotoxic T cell lymphocytes (CD8positive) and activated lymphocytes (CD69 positive) is significantlydecreased in tumors with high Ovr110 expression compared to tumors lowOvr110 expression. Additionally, the median number of infiltrating CD3positive, CD8 positive and CD69 positive lymphocytes is lower comparedto peritumoral CD3 positive, CD8 positive and CD69 positive lymphocytes.These results demonstrate that Ovr110 plays a role in shielding tumorsfrom immune cell surveillance and limiting T cell tumor infiltration.

Summary Ovr110 Expression and T Cell Lymphocyte Invasion

We have demonstrated that Ovr110 is highly expressed in tumors and theexpression of Ovr110 is inversely related to prevalence oftumor-associated CD3, CD8 and CD69 positive lymphocytes. This indicatesthat Ovr110 shields tumor cells from T cell-mediated immunesurveillance, limits tumor infiltration by T cells and preventsapoptosis in tumor cells. The ability to evade immune surveillance is akey virulence factor for tumors which aids in growth, infiltration andmetastases. Additionally, the lack of apoptotic cells, increasedproliferation and invasive properties of the cells examined by IHCdemonstrate that over-expression of Ovr110 by cells increases thetumorgenicity of epithelial cells.

Thus, blocking or inhibiting Ovr110 over-expression or Ovr110 activity(including binding to a receptor) is useful for killing cancer cells,preventing growth of cancer cells, preventing growth/metastases oftumors and shrinking tumors. Anti-Ovr110 compounds such as theantibodies described above bind to cancer cells over-expressing Ovr110,inhibit Ovr110 function and/or prevent inhibition of T cell activationand infiltration into tumors, resulting in an immune response againstthe tumor cells.

Example 4 Deposits Deposit of Cell Lines and DNA

The following hybridoma cell lines were deposited with the American TypeCulture Collection (ATCC), located at 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A., and accorded accession numbers.

The names of the deposited hybridoma cell lines may be shortened forconvenience of reference. E.g. A57.1 corresponds to Ovr110.A57.1. Thesehybridomas correspond to the clones (with their full names) listed inTable 12.

TABLE 12 ATCC deposits Hybridoma ATCC Accession No. Deposit DateOvr110.A57.1 PTA-5180 May 8, 2003 Ovr110.A7.1 PTA-5855 Mar. 11, 2004Ovr110.A72.1 PTA-5856 Mar. 11, 2004 Ovr110.C3.2 PTA-5884 Mar. 23, 2004Ovr110.C6.3 PTA-6266 Oct. 28, 2004 Ovr110.C11.1 PTA-7128 Sep. 30, 2005Ovr110.C12.1 PTA-7129 Sep. 30, 2005

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweendiaDexus, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws. The making of thesedeposits is by no means an admission that deposits are required toenable the invention.

1-32. (canceled) 33: A method of killing head and neck or endometrialcancer cell, comprising contacting the cancer cell with an isolatedanti-Ovr110 antibody, thereby killing the cancer cell. 34: The method ofclaim 33, wherein the the antibody is a monoclonal antibody. 35: Themethod of claim 33, wherein the head and neck cancer is adenoid cysticcarcinoma. 36: The method of claim 35, wherein the localization of theadenoid cystic carcinoma is selected from the group consisting of thetongue, parotid gland, nose, palate, skin, neck, submandibular gland,glottis, sinus, epiglottis, buccal space, nerves, larynx, mouth,pharynx, and cheek. 37: The method of claim 33, wherein the cancer cellis from metastatic head and neck cancer. 38: The method of claim 33,wherein the antibody is an antibody fragment. 39: The method of claim33, wherein the antibody is a humanized antibody. 40: The method ofclaim 33, wherein the antibody is conjugated to a cytotoxic agent. 41:The method of claim 40, wherein the cytotoxic agent is a toxin selectedfrom the group consisting of maytansinoid, ricin, saporin andcalicheamicin. 42: The method of claim 33, wherein the antibody is ahumanized form of the antibody produced by a hybridoma selected from thegroup consisting of ATCC accession number PTA-5180, PTA-5855, PTA-5856,PTA-5884, PTA-6266, PTA-7128 and PTA-7129. 43: The method of claim 40,wherein the cytotoxic agent is selected from the group consisting oftoxins, antibiotics, radioactive isotopes and nucleoytic enzymes. 44: Amethod of alleviating head and neck or endometrial cancer in a mammal,comprising administering a therapeutically effective amount of anisolated anti-Ovr110 antibody to the mammal. 45: The method of claim 44,wherein the antibody is a monoclonal antibody. 46: The method of claim44 wherein the head and neck cancer is adenoid cystic carcinoma. 47: Themethod of claim 44, wherein the antibody is a humanized antibody. 48:The method of claim 44, wherein the antibody is conjugated to acytotoxic agent. 49: The method of claim 48, wherein the cytotoxic agentis a maytansinoid. 50: The method of claim 49, wherein the antibody isadministered in conjunction with at least one chemotherapeutic agent.51: The method of claim 50, wherein the chemotherapeutic agent ispaclitaxel or derivatives thereof. 52: An article of manufacturecomprising a container, a composition contained therein, wherein thecomposition comprises an anti-Ovr110 antibody, and a package insertindicating that the composition can be used to treat head and neck orendometrial cancer. 53: The article of manufacture of claim 52 whereinthe antibody is a monoclonal, humanized or human antibody. 54-63.(canceled) 64: A method for detecting head and neck or endometrialcancer in a subject comprising, (a) combining a bodily fluid sample of asubject with an anti-Ovr110 antibody under conditions suitable forspecific binding of the anti-Ovr110 antibody to Ovr110 in said sample,(b) determining the level of Ovr110 in said sample, (c) comparing thelevel of Ovr110 determined in step (b) to the level of Ovr110 in acontrol, wherein an increase in the level of Ovr110 in the sample fromthe subject as compared to the control is indicative of the presence ofhead and neck and endometrial cancer in the subject. 65: The method ofclaim 64 wherein the antibody is a monoclonal antibody.
 66. (canceled)67: The method of claim 66 wherein the head and neck cancer adenoidcystic carcinoma. 68: The method of claim 64 wherein the control is asample from a subject without head and neck or endometrial cancer.69-75. (canceled) 76: A method for modulating an immune responsecomprising binding Ovr110 with an anti-Ovr110 antibody thereby reducinga suppressed immune function. 77: The method of claim 76 wherein themodulation is an increased immune response. 78: The method of claim 76wherein the modulation is a reduction of suppression of an immuneresponse. 79: The method of claim 76 wherein the immune response isdirected to a cell. 80: The method of claim 79 wherein the cell is acancer cell. 81: The method of claim 80 wherein the cancer cell isselected from the group consisting of head and neck, ovarian,pancreatic, lung, endometrial and breast cancer. 82: The method of claim76 wherein the immune response is increased numbers of lymphocytessurrounding a tumor, increased infiltration of lymphocytes in a tumor,or increased activation of lymphocytes. 83: The method of claim 76wherein the immune function is killing a cancer cell. 84: The method ofclaim 83 wherein the cancer cell is selected from the group consistingof head and neck, ovarian, pancreatic, lung, endometrial and breastcancer. 85: A method for increasing activation of lymphocytes comprisingbinding Ovr110 with an anti-Ovr110 antibody thereby reducing suppressionof lymphocyte activation. 86: The method of claim 85 wherein thelymphocyte is a T cell lymphocyte. 87: The method of claim 85 whereinthe antibody is a monoclonal antibody. 88: The method of claim 76wherein the antibody is a monoclonal antibody. 89: A kit for detectingthe presence of head and neck or endometrial cancer comprising ananti-Ovr110 antibody and purified Ovr110.